Tumor necrosis factor-gamma

ABSTRACT

Human TNF-gamma-alpha and TNF-gamma-beta polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing such polypeptides to inhibit cellular growth, for example in a tumor or cancer, for facilitating wound-healing, to provide resistance against infection, induce inflammatory activities, and stimulating the growth of certain cell types to treat diseases, for example restenosis. Also disclosed are diagnostic methods for detecting a mutation in the TNF-gamma-alpha and TNF-gamma-beta nucleic acid sequences or overexpression of the TNF-gamma-alpha and/or TNF-gamma-beta polypeptides. Antagonists against such polypeptides and their use as a therapeutic to treat cachexia, septic shock, cerebral malaria, inflammation, arthritis and graft-rejection are also disclosed.

RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C.§119(e) based on U.S. Provisional Application Serial Nos. 60/278,449filed Mar. 26, 2001 and 60/216,879 filed Jul. 7, 2000. This applicationis also a continuation-in-part of U.S. Utility patent application Ser.No. 09/559,290 filed Apr. 27, 2000 which claims the benefit of priorityunder 35 U.S.C. §119(e) based on U.S. Provisional Application SerialNos. 60/180,908 filed Feb. 8, 2000, 60/134,067 filed May 13, 1999,60/132,227 filed May 3, 1999, and 60/131,963 filed Apr. 30, 1999. U.S.Utility patent application Ser. No. 09/559,290 is also acontinuation-in-part of U.S. Utility patent application Ser. No.09/246,129 filed Feb. 8, 1999 which claims the benefit of priority under35 U.S.C. §119(e) based on U.S. Provisional Application Serial No.60/074,047 filed Feb. 9, 1998. U.S. Utility patent application Ser. No.09/246,129 is also a continuation-in-part of U.S. Utility patentapplication Ser. No. 09/131,237 filed Aug. 7, 1998 which is acontinuation-in-part of U.S. Utility patent application Ser. No.09/005,020 filed Jan. 9, 1998, now abandoned, which is acontinuation-in-part of U.S. Utility patent application Ser. No.08/461,246 filed Jun. 5, 1995, now abandoned, which is a continuation inpart of PCT/US94/12880 filed Nov. 7, 1994.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention has been identified as a new member of the tumornecrosis factor family and is hereinafter referred to as“TNF-gamma-alpha”. The invention also relates to a protein encoded by asplice variant of the gene encoding TNF-gamma-alpha which is hereinafterreferred to as “TNF-gamma-beta”. The invention also relates toinhibiting the action of such polypeptides.

BACKGROUND OF THE INVENTION

[0003] Human tumor necrosis factors-alpha (TNF-alpha) and beta (TNF-betaor lymphotoxin) are related members of a broad class of polypeptidemediators, which includes the interferons, interleukins and growthfactors, collectively called cytokines (Beutler, B. and Cerami, A.,Annu. Rev. Immunol., 7:625-655 (1989)).

[0004] Tumor necrosis factor (TNF-alpha and TNF-beta) was originallydiscovered as a result of its anti-tumor activity, however, now it isrecognized as a pleiotropic cytokine playing important roles in immuneregulation and inflammation. To date, there are eight known members ofthe TNF-related cytokine family, TNF-alpha, TNF-beta (lymphotoxin(LT)-alpha), LT-beta, and ligands for the Fas, CD30, CD27, CD40 and4-1BB receptors. These proteins have conserved C-terminal sequences andvariable N-terminal sequences which are often used as membrane anchors,with the exception of TNF-beta. Both TNF-alpha and TNF-beta function ashomotrimers when they bind to TNF receptors.

[0005] TNF is produced by a number of cell types, including monocytes,fibroblasts, T-cells, natural killer (NK) cells and predominately byactivated machrophages. TNF-alpha has been reported to have a role inthe rapid necrosis of tumors, immunostimulation, autoimmune disease,graft rejection, resistance to parasites, producing an anti-viralresponse, septic shock, growth regulation, vascular endothelium effectsand metabolic effects. TNF-alpha also triggers endothelial cells tosecrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 topromote cell proliferation. In addition, TNF-alpha up-regulates variouscell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1. TNF-alphaand Fas ligand have also been shown to induce programmed cell death.

[0006] The first step in the induction of the various cellular responsesmediated by TNF or LT is their binding to specific cell surfacereceptors. Two distinct TNF receptors of approximately 55-KDa (TNF-R1)and 75-KDa (TNF-R2) have been identified (Hohman, H. P. et al., J. Biol.Chem., 264:14927-14934 (1989)), and human and mouse cDNAs correspondingto both receptor types have been isolated and characterized (Loetscher,H. et al., Cell, 61:351 (1990)). Both TNF-Rs share the typical structureof cell surface receptors including extracellular, transmembrane andintracellular regions.

[0007] The endothelium, which under physiological conditions is mostly aquiescent tissue (Denekamp, J. Cancer Metas. Rev. 9:267-282 (1990)),plays an essential role in the maintenance of vascular homeostasis andpermeability. Endothelial cells are actively involved in inflammation,cell adhesion, coagulation, thrombosis, fibrinolysis, and angiogenesis.During angiogenesis, endothelial cells proliferate, invade into stroma,migrate toward the source of an angiogenesis stimulus, such as cancercells, interact with perivascular cells and stromal cells, andeventually, form capillary vessels linking the tumor tissue to thecirculatory system (Folkman, J. Nature Med. 1:27-31 (1995)). Althoughthe complex mechanism that regulates angiogenesis is yet to be fullyunderstood, it is becoming clear that the initiation or termination ofthe process is a result of a balance between positive and negativefactors.

[0008] A number of angiogenic factors, often markedly upregulated intumor tissues, have been described. These include several members of thefibroblast growth factor (FGF) family, such as FGF-1, FGF-2, and thoseof the vascular endothelial cell growth factor (VEGF) family and thereceptors for all of these molecules (Gimenez-Gallego, G, et al.,Science 230:1385-1388 (1985); Schweigerer, L., et al., Nature325:257-259 (1987); Leung, D. W., et al., Science 246:1306-1309 (1989);Burrus, L. W. and Olwin, B. B. J. Biol. Chem. 264:18647-18653 (1989);Wennstrom, S., et al., Growth Factors 4:197-208 (1991); Terman, B. I.,et al., Biochem. Biophys. Res. Comm. 187:1579-1586 (1992); de Vries, C.,et al., Science 255:989-991 (1992)). Likewise, several inhibitors ofangiogenesis have also been reported, including thrombospondin,angiostatin, endostatin, and platelet factor-4 (Good, D. J., et al.,Proc. Natl. Acad. Sci. USA 87:6623-6628 (1990); O'Reilly, M. S., et al.,Cell 79:315-328 (1994); O'Reilly, M. S., et al., Cell 88:277-285 (1997);Maione, T. E., et al., Science 247:77-79 (1990)). It is apparent thatnormal angiogenesis is promptly activated when needed, and swiftlyterminated when no longer required. However, pathological angiogenesis,once initiated, is often prolonged and often difficult to stop. This mayindicate that a negative regulatory mechanism normally functioning ismissing or suppressed in a pathological angiogenic process. It isconceivable that endothelial cells may produce autocrine factors tosuppress an angiogenesis process or maintain the quiescence of a maturevasculature.

[0009] The polypeptide of the present invention has been identified as anovel member of the TNF family based on structural, amino acid sequencehomology, and functional similarities, for example, TNF-gamma is apro-inflammatory protein. Further, the TNF-gamma polypeptide of thepresent invention is a negative regulator of angiogenesis and ofendothelial cell growth. There is a need for polypeptides that functionin this manner, since disturbances of such regulation may be involved indisorders relating to angiogenesis, hemostasis, tumor metastasis,cellular migration, and cancers of many systems. Therefore, there is aneed for identification and characterization of such human polypeptideswhich can play a role in detecting, preventing, ameliorating orcorrecting such disorders.

SUMMARY OF THE INVENTION

[0010] In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is TNF-gamma-alpha, and anovel mature polypeptide which is TNF-gamma-beta, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

[0011] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding humanTNF-gamma-alpha or TNF-gamma-beta, including mRNAs, DNAs, cDNAs, genomicDNAs as well as analogs and biologically active and diagnostically ortherapeutically useful fragments and derivatives thereof.

[0012] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of theTNF-gamma-alpha polypeptide having the complete amino acid sequenceshown in SEQ ID NO:2 or the complete amino acid sequence encoded by thecDNA clone HUVE091 deposited as plasmid DNA as ATCC Deposit Number 75927at the American Type Culture Collection (“ATCC”) on Oct. 26, 1994. TheATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209,USA. The nucleotide sequence determined by sequencing the depositedTNF-gamma-alpha clone, which is shown in FIGS. 1A and 1B (SEQ ID NO:1),contains an open reading frame encoding a complete polypeptide of 174amino acid residues, including an initiation codon encoding anN-terminal methionine at nucleotide positions 783-785, and a predictedmolecular weight of about 20,132 Da.

[0013] The present invention also provides isolated nucleic acidmolecules comprising a polynucleotide encoding at least a portion of theTNF-gamma-beta polypeptide having the complete amino acid sequence shownin SEQ ID NO:20 or the complete amino acid sequence encoded by the cDNAclone HEMCZ56 deposited as plasmid DNA as ATCC Deposit Number 203055 onJul. 9, 1998. The nucleotide sequence determined by sequencing thedeposited TNF-gamma-beta clone, which is shown in FIGS. 20A and B (SEQID NO:20), contains an open reading frame encoding a completepolypeptide of 251 amino acid residues, including an initiation codonencoding an N-terminal methionine at nucleotide positions 1-3, and apredicted molecular weight of about 28,089 Da.

[0014] Thus, in one embodiment the invention provides an isolatednucleic acid molecule comprising a polynucleotide having a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence encoding the TNF-gamma-alpha polypeptide having the completeamino acid sequence in SEQ ID NO:2 (i.e., positions −27 to 147 of SEQ IDNO:2, (b) a nucleotide sequence encoding the TNF-gamma-alpha polypeptidehaving the complete amino acid sequence in SEQ ID NO:2 excepting theN-terminal methionine (i.e., positions −26 to 147 of SEQ ID NO:2); (c) anucleotide sequence encoding the mature TNF-gamma-alpha polypeptidehaving the amino acid sequence in SEQ ID NO:2 shown as positions 1 to147 of SEQ ID NO:2; (d) a nucleotide sequence encoding theTNF-gamma-alpha polypeptide having the complete amino acid sequenceencoded by the cDNA clone HUVEO91 contained in ATCC Deposit No. 75927;(e) a nucleotide sequence encoding the TNF-gamma-alpha polypeptidehaving the complete amino acid sequence excepting the N-terminalmethionine encoded by the cDNA clone HUVEO91 contained in ATCC DepositNo. 75927; (f) a nucleotide sequence encoding the mature TNF-gamma-alphapolypeptide having the amino acid sequence encoded by the cDNA cloneHUVEO91 contained in ATCC Deposit No. 75927; and (g) a nucleotidesequence complementary to any of the nucleotide sequences in (a), (b),(c), (d), (e) or (f), above.

[0015] In another embodiment, the invention provides an isolated nucleicacid molecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the TNF-gamma-beta polypeptide having the complete amino acidsequence in SEQ ID NO:20 (i.e., positions 1 to 251 of SEQ ID NO:20); (b)a nucleotide sequence encoding the TNF-gamma-beta polypeptide having thecomplete amino acid sequence in SEQ ID NO:20 excepting the N-terminalmethionine (i.e., positions 2 to 251 of SEQ ID NO:20); (c) a nucleotidesequence encoding the mature TNF-gamma-beta polypeptide having the aminoacid sequence in SEQ ID NO:20 shown as positions 60 to 251 of SEQ IDNO:20; (d) a nucleotide sequence encoding the mature TNF-gamma-betapolypeptide having the amino acid sequence in SEQ ID NO:20 shown aspositions 62 to 251 of SEQ ID NO:20; (e) a nucleotide sequence encodingthe mature TNF-gamma-beta polypeptide having the amino acid sequence inSEQ ID NO:20 shown as positions 72 to 251 of SEQ ID NO:20; (f) anucleotide sequence encoding the TNF-gamma-beta polypeptide having thecomplete amino acid encoded by the cDNA clone HEMCZ56 contained in ATCCDeposit No. 203055; (g) a nucleotide sequence encoding theTNF-gamma-beta polypeptide having the complete amino acid sequenceexcepting the N-terminal methionine encoded by the cDNA clone HEMCZ56contained in ATCC Deposit No. 203055; (h) a nucleotide sequence encodingthe mature TNF-gamma-beta polypeptide having the amino acid sequenceencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;and (i) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g) or (h), above.

[0016] In accordance with all aspects of the invention, the term“TNF-gamma” refers to TNF-gamma-alpha and/or TNF-gamma-beta.

[0017] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 80%, 85% or 90% identical, and more preferably atleast 92%, 94%, 95%, 96%, 97%, 98% or 99% identical, to any of theTNF-gamma-alpha or TNF-gamma-beta nucleotide sequences in (a), (b), (c),(d), (e), (f), (g), (h) or (i), above, or a polynucleotide whichhybridizes under stringent hybridization conditions to a TNF-gamma-alphaor TNF-gamma-beta polynucleotide in (a), (b), (c), (d), (e), (f), (g) or(h), above, a fragment thereof (such as, for example, fragmentsdescribed herein), or the complementary strand thereto. Thispolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues. Anadditional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion (i.e., a fragment)of a TNF-gamma-alpha or TNF-gamma-beta polypeptide having an amino acidsequence in (a), (b), (c), (d), (e), (f), (g) or (h), above. A furtherembodiment of the invention relates to an isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of aTNF-gamma polypeptide having an amino acid sequence which contains atleast one amino acid substitution, but not more than 50 amino acidsubstitutions, even more preferably, not more than 40 amino acidsubstitutions, still more preferably, not more than 30 amino acidsubstitutions, and still even more preferably, not more than 20 aminoacid substitutions. Of course, in order of ever-increasing preference,it is highly preferable for a polynucleotide which encodes the aminoacid sequence of a TNF-gamma polypeptide to have an amino acid sequencewhich contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. Conservative substitutions are preferable.

[0018] The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of TNF-gamma polypeptides or peptides by recombinanttechniques.

[0019] In accordance with a further aspect of the present invention,there is provided a process for producing such polypeptide byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a human TNF-gamma nucleic acidsequence, under conditions promoting expression of said protein andsubsequent recovery of said protein.

[0020] The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-alphapolypeptide having the complete amino acid sequence shown in SEQ ID NO:2(i.e., positions −27 to 147 of SEQ ID NO:2); (b) the amino acid sequenceof the full-length TNF-gamma-alpha polypeptide having the complete aminoacid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine(i.e., positions −26 to 147 of SEQ ID NO:2); (c) the amino acid sequenceof the predicted mature TNF-gamma-alpha polypeptide having the aminoacid sequence at positions 1-147 in SEQ ID NO:2; (d) the complete aminoacid sequence encoded by the cDNA clone HUVEO91 contained in the ATCCDeposit No. 75927; (e) the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone HUVEO91 contained in theATCC Deposit No. 75927; (f) the complete amino acid sequence of thepredicted mature TNF-gamma polypeptide encoded by the cDNA clone HUVEO91contained in the ATCC Deposit No. 75927; and (g) fragments of thepolypeptide of (a), (b), (c), (d), (e), or (f). The polypeptides of thepresent invention also include polypeptides having an amino acidsequence at least 80% or 85% identical, more preferably at least 90%,92% or 94% identical, and still more preferably 95%, 96%, 97%, 98% or99% identical to those described in (a), (b), (c), (d), (e) (f), or (g)above, as well as polypeptides having an amino acid sequence with atleast 90% similarity, and more preferably at least 95% similarity, tothose above. Additional embodiments of the invention relates to apolypeptide which comprises the amino acid sequence of anepitope-bearing portion of a TNF-gamma-alpha polypeptide having an aminoacid sequence described in (a), (b), (c), (d), (e), (f), or (g) above.Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a TNF-gamma-alpha polypeptide of theinvention include portions of such polypeptides with at least six orseven, preferably at least nine, and more preferably at least about 30amino acids to about 50 amino acids, although epitope-bearingpolypeptides of any length up to and including the entire amino acidsequence of a polypeptide of the invention described above also areincluded in the invention.

[0021] The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-betapolypeptide having the complete amino acid sequence shown in SEQ IDNO:20 (i.e., positions 1 to 251 of SEQ ID NO:20); (b) the amino acidsequence of the full-length TNF-gamma-beta polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:20 excepting theN-terminal methionine (i.e., positions 2 to 251 of SEQ ID NO:20); (c)the amino acid sequence of the predicted mature TNF-gamma-betapolypeptide having the amino acid sequence at positions 60-251 in SEQ IDNO:20; (d) the amino acid sequence of the predicted matureTNF-gamma-beta polypeptide having the amino acid sequence at positions62-251 in SEQ ID NO:20; (e) the amino acid sequence of the predictedmature TNF-gamma-beta polypeptide having the amino acid sequence atpositions 72-251 in SEQ ID NO:20; (f) the complete amino acid sequenceencoded by the cDNA clone HEMCZ56 contained in the ATCC Deposit No.203055; (g) the complete amino acid sequence excepting the N-terminalmethionine encoded by the cDNA clone HEMCZ56 contained in the ATCCDeposit No. 203055; (h) the complete amino acid sequence of thepredicted mature TNF-gamma polypeptide encoded by the cDNA clonecontained in the ATCC Deposit No. 203055; and (i) fragments of thepolypeptide of (a), (b), (c), (d), (e), (f), (g) or (h), above. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% or 85% identical, more preferably atleast 90%, 92% or 94% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e)(f), (g) or (h), above, as well as polypeptides having an amino acidsequence with at least 90% similarity, and more preferably at least 95%similarity, to those above. Additional embodiments of the inventionrelates to a polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a TNF-gamma-beta polypeptide having an aminoacid sequence described in (a), (b), (c), (d), (e), (f), (g) or (h),above. Peptides or polypeptides having the amino acid sequence of anepitope-bearing portion of a TNF-gamma-beta polypeptide of the inventioninclude portions of such polypeptides with at least six or seven,preferably at least nine, and more preferably at least about 30 aminoacids to about 50 amino acids, although epitope-bearing polypeptides ofany length up to and including the entire amino acid sequence of apolypeptide of the invention described above also are included in theinvention.

[0022] A further embodiment of the invention relates to a polypeptidewhich comprises the amino acid sequence of a TNF-gamma polypeptidehaving an amino acid sequence which contains at least one amino acidsubstitution, but not more than 50 amino acid substitutions, even morepreferably, not more than 40 amino acid substitutions, still morepreferably, not more than 30 amino acid substitutions, and still evenmore preferably, not more than 20 amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of a TNF-gamma polypeptide, which contains atleast one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. In specific embodiments, the number of additions,substitutions, and/or deletions in the amino acid sequence of FIGS. 1Aand 1B, FIGS. 20A and B, or fragments thereof (e.g., the extracellulardomain and/or other fragments described herein), is 1-5, 5-10, 5-25,5-50, 10-50 or 50-150, conservative amino acid substitutions arepreferable.

[0023] In another embodiment, the invention provides an isolatedantibody that binds specifically to a TNF-gamma polypeptide having anamino acid sequence described above. The invention further providesmethods for isolating antibodies that bind specifically to a TNF-gammapolypeptide having an amino acid sequence as described herein. Suchantibodies are useful diagnostically or therapeutically as describedbelow. In preferred embodiments, neutralizing or antagonisticanti-TNF-gamma-alpha and/or TNF-gamma-beta antibodies may be used totreat, prevent or diagnose, inflammatory diseases (e.g., inflammatorybowel disease, encephalitis) and immune disorders, especially T-cellmediated immune disorders, including but not limited to autoimmunediseases (e.g., systemic lupus erythematosus, arthritis, multiplesclerosis).

[0024] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing polynucleotides and/orpolypeptides of the invention as well as agonists and antagoniststhereof, and for therapeutic purposes, which include, but are notlimited to, wound healing, to inhibit tumor proliferation, to provideresistance to parasites, bacteria and viruses, to induce inflammatoryactivities, to induce proliferation of endothelial cells and certainhematopoietic cells, to treat, prevent, diagnose, and/or detectrestenosis and to prevent certain autoimmune diseases.

[0025] In accordance with yet a further aspect of the present invention,there are also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to humanTNF-gamma sequences.

[0026] In accordance with another aspect of the present invention, thereare provided TNF-gamma agonists which mimic TNF-gamma and binds to theTNF-gamma receptors to elicit TNF-gamma type responses.

[0027] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, to preventseptic shock, inflammation, cerebral malaria, activation of the HIVvirus, graft rejection, bone resorption and cachexia.

[0028] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting diseases related tothe under-expression and over-expression of the TNF-gamma polypeptideand nucleic acid sequences encoding such polypeptide.

[0029] In a further aspect of the invention, TNF-gamma may be used totreat, prevent, diagnose, and/or detect rheumatoid arthritis (RA) byinhibiting the increase in angiogensis or the increase in endothelialcell proliferation required to sustain an invading pannus in bone andcartilage as is often observed in RA.

[0030] In a further aspect of the invention, TNF-gamma may be used totreat, prevent, diagnose, and/or detect diseases or conditionsincluding, but not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

[0031] In yet another aspect, the TNF-gamma may bind to a cell surfaceprotein which also functions as a viral receptor or coreceptor. Thus,TNF-gamma, or agonists or antagonists thereof, may be used to regulateviral infectivity at the level of viral binding or interaction with theTNF-gamma receptor or coreceptor or during the process of viralinternalization or entry into the cell.

[0032] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

[0033] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0034]FIGS. 1A and 1B illustrate the cDNA (SEQ ID NO:1) andcorresponding deduced amino acid sequence (SEQ ID NO:2) of thepolypeptide of TNF-gamma-alpha of the present invention. The initial 27amino acids (underlined) are the putative leader sequence. The standardone-letter abbreviations for amino acids are used. Potentialasparagine-linked glycosylation sites are marked in FIGS. 1A and 1B witha bolded asparagine symbol (N) in the TNF-gamma-alpha amino acidsequence and a bolded pound sign (#) above the first nucleotide encodingthat asparagine residue in the TNF-gamma-alpha nucleotide sequence.Potential N-linked glycosylation sequences are found at the followinglocations in the TNF-gamma-alpha amino acid sequence: N-29 through N-32(N-29, Y-30, T-31, N-32) and N-125 through D-128 (N-125, V-126, S-127,D-128). Potential Protein Kinase C (PKC) phosphorylation sites are alsomarked in FIGS. 1A and 1B with a bolded threonine symbol (T) in theTNF-gamma-alpha amino acid sequence and an asterisk (*) above the firstnucleotide encoding that threonine residue in the TNF-gamma-alphanucleotide sequence. Potential PKC phosphorylation sequences are foundat the following locations in the TNF-gamma-alpha amino acid sequence:T-32 through K-34 (T-32, N-33, K-34) and T-50 through R-52 (T-50, F-51,R-52). Potential Casein Kinase II (CK2) phosphorylation sites are alsomarked in FIGS. 1A and 1B with a bolded serine or threonine symbol (S orT) in the TNF-gamma-alpha amino acid sequence and an asterisk (*) abovethe first nucleotide encoding the appropriate serine or threonineresidue in the TNF-gamma-alpha nucleotide sequence. Potential CK2phosphorylation sequences are found at the following locations in theTNF-gamma-alpha amino acid sequence: S-83 through E-86 (S-83, Y-84,P-85, E-86); S-96 through E-99 (S-96, V-97, C-98, E-99); S-115 throughE-118 (S-115, L-116, Q-117, E-118); S-130 through D-133 (S-130, L-131,V-132, D-133); and T-135 through D-138 (T-135, K-136, E-137, D-138).Potential myristylation sites are also marked in FIGS. 1A and 1B with adouble underline in the TNF-gamma-alpha amino acid sequence. Potentialmyristylation sequences are found at the following locations in theTNF-gamma-alpha amino acid sequence: G-20 through K-25 (G-20, L-21,A-22, F-23, T-24, K-25) and G-111 through L-116 (G-111, A-112, M-113,F-114, S-115, L-116).

[0035]FIGS. 2A, 2B, and 2C illustrate an amino acid sequence alignmentbetween TNF-gamma-alpha (SEQ ID NO:2) and other members of the TNFfamily including human TNF-alpha (GenBank No. Z15026; SEQ ID NO:3),human TNF-beta (GenBank No. Z15026; SEQ ID NO:4), human lymphotoxin-beta(LTbeta; GenBank No. L11016; SEQ ID NO:5), and rat Fas Ligand (FASL:GenBank No. U034070; SEQ ID NO:6). TNF-gamma contains the conservedamino acid residues of the TNF family as shown by the boxed and shadedareas. The aligned molecules are presented in their entirety as FIGS.2A, 2B, and 2C.

[0036]FIG. 3A is an RNA blot analysis showing the human tissues whereTNF-gamma is expressed. RNA from the tissues indicated were probed withlabeled TNF-gamma cDNA. TNF-gamma-alpha mRNA exists predominantly in thekidney since FIG. 3A shows a distinct band. Other lanes seem to showstrong hybridization, however, these are actually non-specific smears.

[0037]FIG. 3B is an RNA blot analysis showing that TNF-gamma isexpressed predominantly in HUVEC cells (human umbilical vein endothelialcells; lane 9). Lane 6 and lane 8 are non-specific smears. RNA from thecell lines indicated were probed with labeled TNF-gamma-alpha cDNA. Lane1 is CAMA1 (breast cancer); lane 2 AN3CA (uterine cancer); lane 3,SK.UT.1 (uterine cancer); lane 4, MG63 (osteoblastoma); lane 5, HOS(osteoblastoma); lane 6, MCF7 (breast cancer); lane 7, OVCAR-3 (ovariancancer); lane 8, CAOV-3 (ovarian cancer); lane 9, HUVEC; lane 10, AOSMIC(smooth muscle); lane 11, foreskin fibroblast.

[0038]FIG. 4 illustrates the relative expression of TNF-gamma inproliferating or quiescent endothelial cells. The TNF-gamma mRNA levelsin cultured HUVEC cells were determined by Northern blotting analysis.Identical amounts of total RNA (15 μg) were loaded on each lane, asindicated by the intensity of b-actin. The signal which corresponds toTNF-gamma is designated “VEGI”. Total RNA was prepared at the indicatedtime point (days post-seeding). The number of cells in each cultureflask was determined simultaneously. The experiment was carried out induplicate. Cells were seeded at 125,00 cells per flask (T-25).

[0039]FIG. 5 is a photograph of a polyacrylamide gel electrophoresisanalysis of TNF-gamma protein. TNF-gamma was produced by bacterialexpression and purified as described in Example 1.

[0040]FIG. 6 is a photograph of a gel showing the relative purity andmobility of baculovirus-expressed TNF-gamma. The expression andpurification of TNF-gamma using the baculovirus system is described inExample 2.

[0041]FIG. 7A consists of photographs of WEHI 164 cells which areuntreated (FIG. 7Aa) and after exposure to TNF-alpha (FIG. 7Ab),TNF-gamma (FIG. 7Ac), and TNF-beta (FIG. 7Ad). Cells which have anelongated non-round morphology have been lysed. The various TNFmolecules were added at a concentration of approximately 0.5 μg/ml.Photographs were taken 72 hours after addition of the various TNFmolecules.

[0042]FIG. 7B illustrates the ability of TNF-gamma (FIG. 7Bc) incomparison to TNF-alpha (FIG. 7Ba) and TNF-beta (FIG. 7Bb) to inhibitWEHI 164 cell growth.

[0043]FIG. 8 illustrates the ability of recombinant TNF-alpha (FIG. 8B),TNF-beta (FIG. 8D), and TNF-gamma (FIG. 8C) to induce morphologicalchange in L929 cells with respect to untreated L929 cells (FIG. 8A). Themorphology change is indicated by dark round cells. Cells were treatedwith the various recombinant TNF molecules (produced in E. coli) atapproximately 0.5 μg/ml. The photographs were taken 72 hours after theaddition of the various TNF molecules. The morphology change indicatesthat the cells have been killed.

[0044]FIG. 9 is a graphical illustration of the effect of TNF-gamma(FIG. 9C), TNF-alpha (FIG. 9A), and TNF-beta (FIG. 9B) on venousendothelial cells. Cell proliferation after venous endothelial cellswere treated with commercially available TNF-alpha and TNF-beta and E.coli produced TNF-gamma was quantified using an MTS assay.

[0045]FIG. 10 shows the effect of TNF-gamma on the proliferation ofendothelial cell and breast cancer cells. The number of cells areplotted against TNF-gamma concentration as indicated (TNF-gamma isdesignated “VEGI” in this figure). Inhibition of the growth of adultbovine aortic endothelial (ABAE) cells (dark circles), but not that ofMDA-MB-231 (dark triangles) or MDA-MB-435 (open circles) cells, isshown. The cells were seeded at 2×10³ cells/well in triplicate in24-well plates. The ABAE cell culture media contained IMEM (LifeTechnologies, Inc., Rockville, Md.) supplemented with 10% FCS and (1ng/ml) FGF-2. The cultures were maintained at 37° C., 5% CO₂, for 6days. The cells were then trypsinized, and the number of cellsdetermined by using a Coulter counter. One-fifth of the total number ofrecovered ABAE cells is shown in order to normalize the comparison withthe MDA-MB-231 and MDA-MB-435 cells.

[0046]FIG. 11 is a photograph of HL60 cells, with control (FIG. 11A)showing the HL60 cells being spread apart; TNF-alpha (FIG. 11B) andTNF-gamma (FIG. 11C) induce cell adhesion and cell-cell contact asillustrated by the cells adhering together in the lower right.

[0047]FIG. 12 illustrates the ability of recombinant TNF-gamma(represented by squares), TNF-alpha (represented by circles), andTNF-beta (represented by triangles) to induce WEHI 164 cell death. Celldeath is inversely proportional to the ratio of absorbance at 405 nm tothat at 490 nm).

[0048]FIG. 13 illustrates that TNF-gamma does not significantly bind totwo known soluble TNF receptors, namely sTNF RI (p55; solid bars) andsTNF RII (p75; hatched bars).

[0049]FIG. 14 demonstrates the effect of TNF-gamma on the ability ofABAE cells to form capillary-like tubes on collagen gels. The ability ofrecombinant TNF-gamma (residues 12-147 as shown in SEQ ID NO:2 anddesignated “VEGI” in this figure) to inhibit the formation ofcapillary-like tubes by ABAE cells is shown. The p-values (t-test) givenabove the columns are obtained by comparing the extent of thecapillary-like tube formation by ABAE cells in the presence of variousconcentrations of TNF-gamma, as indicated, to that when TNF-gamma isabsent from the culture media.

[0050]FIG. 15 shows the inhibition of angiogenesis in collagen gelsplaced on chicken embryonic chorioallantoic membrane (CAM) by TNF-gamma.The growth of new capillary vessels into collagen gel pellets placed onthe CAM was induced by either FGF-2 (100 ng) or VEGF (250 ng). Theextent of angiogenesis in the gels was determined by evaluation of thefluorescence intensity of FITC-dextran injected into the CAMcirculation. Inhibition of the capillary vessel growth by therecombinant TNF-gamma (designated “VEGI” in this figure), as indicatedby a lower value than 100, is shown. The experiment was carried out intriplicate.

[0051]FIG. 16 illustrates the inhibition of growth of human breastcancer xenograft tumors in athymic nude mice by TNF-gamma. Mixtures ofTNF-gamma-overexpressing or vector-transfected CHO cells (5×10⁶ cellsper injection) and human breast cancer cells (1×10⁶ cells per injection)were injected into the mammary fat pads of the nude mice. Tumor sizes(area) were monitored following injection. The sizes of the MDA-MB-231xenograft tumors (mm²) were plotted as a function of dayspost-inoculation (FIG. 16A). The sizes of the MDA-MB-435 xenografttumors (mm²) were plotted as a function of days post-inoculation (FIG.16B). Open circles represent values of tumors co-inoculated withvector-transfected CHO cells, whereas closed circles represent values oftumors co-inoculated with TNF-gamma-transfected CHO cells.

[0052]FIG. 17 shows an analysis of the TNF-gamma-alpha amino acidsequence (SEQ ID NO:2). Alpha, beta, turn and coil regions;hydrophilicity and hydrophobicity; amphipathic regions; flexibleregions; antigenic index and surface probability are shown, as predictedusing the default parameters of the recited computer programs. In the“Antigenic Index or Jameson-Wolf” graph, the positive peaks indicatelocations of the highly antigenic regions of the TNF-gamma protein,i.e., regions from which epitope-bearing peptides of the invention canbe obtained.

[0053]FIGS. 18A, 18B, 18C, and 18D show an alignment of the nucleotidesequences of TNF-gamma-alpha (SEQ ID NO:1) and TNF-gamma-beta (SEQ IDNO:19) constructed by using the computer program BESTFIT set at defaultparameters.

[0054]FIG. 19 shows an alignment of the amino acid sequences ofTNF-gamma-alpha (SEQ ID NO:2) and TNF-gamma-beta (SEQ ID NO:20)constructed using the default parameters of the computer programBESTFIT.

[0055]FIGS. 20A and 20B illustrate the cDNA (SEQ ID NO:19) andcorresponding deduced amino acid sequence (SEQ ID NO:20) of thepolypeptide of the TNF-gamma-beta of the present invention. The standardone-letter abbreviations for amino acids are used. In one embodiment,amino acids methionine-1 to tryptophan-35 comprise the predictedintracellular domain. Amino acid residues alanine-36 through alanine-61(underlined) comprise the putative transmembrane sequence. Amino acidresidues glutamine-62 through leucine-251 (underlined) comprise theputative extracellular domain. In another embodiment, amino acidsmethionine-1 to tryptophan-35 comprise the predicted intracellulardomain; amino acid residues alanine-36 through leucine-59 comprise theputative transmembrane sequence; and amino acid residues arginine-60through leucine-251 comprise the putative extracellular domain.Potential asparagine-linked glycosylation sites are marked in FIGS. 20Aand B with a bolded asparagine symbol (N) in the TNF-gamma-beta aminoacid sequence and a bolded pound sign (#) above the first nucleotideencoding that asparagine residue in the TNF-gamma-alpha nucleotidesequence. Potential N-linked glycosylation sequences are found at thefollowing locations in the TNF-gamma-beta amino acid sequence: N-133through N-136 (N-133, Y-134, T-135, N-136) and N-229 through D-232(N-229, V-230, S-231, D-232). Potential Protein Kinase C (PKC)phosphorylation sites are also marked in FIGS. 20A and B with a boldedserine or threonine symbol (S or T) in the TNF-gamma-beta amino acidsequence and an asterisk (*) above the first nucleotide encoding thatthreonine residue in the TNF-gamma-beta nucleotide sequence. PotentialPKC phosphorylation sequences are found at the following locations inthe TNF-gamma-beta amino acid sequence: S-23 through R-25 (S-23, C-24,R-25); S-32 through R-34 (S-32, A-33, R-34); T-135 through K-137 (T-135,N-136, K-137); and T-154 through R-156 (T-154, F-155, R-156). PotentialCasein Kinase II (CK2) phosphorylation sites are also marked in FIGS.20A and B with a bolded serine or threonine symbol (S or T) in theTNF-gamma-beta amino acid sequence and an asterisk (*) above the firstnucleotide encoding the appropriate serine or threonine residue in theTNF-gamma-beta nucleotide sequence. Potential CK2 phosphorylationsequences are found at the following locations in the TNF-gamma-betaamino acid sequence: S-8 through E-11 (S-8, F-9, G-10, E-11); S-187through E-190 (S-187, Y-188, P-189, E-190); S-200 through E-203 (S-200,V-201, C-202, E-203); S-219 through E-222 (S-219, L-220, Q-221, E-222);S-234 through D-237 (S-234, L-235, V-236, D-237); and T-239 throughD-242 (T-239, K-240, E-241, D-242). Potential myristylation sites arealso marked in FIGS. 20A and B with a double underline in theTNF-gamma-beta amino acid sequence. Potential myristylation sequencesare found at the following locations in the TNF-gamma-beta amino acidsequence: G-6 through E-11 (G-6, L-7, S-8, F-9, G-10, E-11); G-124through G-129 (G-124, L-125, A-126, F-127, T-128, K-129); and G-215through L-220 (G-215, A-216, M-217, F-218, S-219, L-220).

DETAILED DESCRIPTION

[0056] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a TNF-gamma-alpha polypeptidehaving the amino acid sequence shown in FIGS. 1A and B (SEQ ID NO:2),which was determined by sequencing a cloned cDNA (HUVEO91). As shown inFIGS. 2A-2C, the TNF-gamma-alpha polypeptide of the present inventionshares sequence homology with human TNF-alpha (SEQ ID NO:3), TNF-beta(SEQ ID NO:4), human lymphotoxin-beta (SEQ ID NO:5) and FAS ligand (SEQID NO:6). The TNF-gamma-alpha of the invention functions at least in theinhibition of angiogenesis, as an anti-tumor cytokine-like molecule, asa treatment for arthritis by the inhibition of angiogenesis and/orendothelial cell proliferation associated with invading pannus in boneand cartilage, as an inducer of NF-kappaB and c-Jun kinase (JNK), aninducer of cell adhesion, and as an inducer of apoptosis (See Examples,particularly Examples 12-15). The nucleotide sequence shown in SEQ IDNO:1 was obtained by sequencing a cDNA clone (HUVEO91), which wasdeposited on Oct. 26, 1994 at the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209, and givenaccession number 75927. The deposited plasmid is contained inpBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.). Furthercharacterization of the protein encoded by clone HUVEO91 is presented incopending U. S. Provisional Application Serial No. 60/074,047, filedFeb. 9, 1998; the entire disclosure of which is hereby incorporated byreference.

[0057] The present invention also provides isolated nucleic acidmolecules comprising a polynucleotide (SEQ ID NO:19) encoding aTNF-gamma-beta polypeptide having the amino acid sequence shown in FIGS.20A and B (SEQ ID NO:20), which was determined by sequencing a clonedcDNA (HEMCZ56). The TNF-gamma-beta polypeptide is a splice variant ofthe TNF-gamma-alpha polypeptide disclosed herein. The nucleotidesequence shown in SEQ ID NO:19 was obtained by sequencing a cDNA clone(HEMCZ56), which was deposited on Jul. 9, 1998 at the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, and given accession number 203055. The deposited plasmid iscontained in pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.).

Nucleic Acid Molecules

[0058] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.,Foster City, Calif.), and all amino acid sequences of polypeptidesencoded by DNA molecules determined herein were predicted by translationof a DNA sequence determined as above. Therefore, as is known in the artfor any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart. As is also known in the art, a single insertion or deletion in adetermined nucleotide sequence compared to the actual sequence willcause a frame shift in translation of the nucleotide sequence such thatthe predicted amino acid sequence encoded by a determined nucleotidesequence will be completely different from the amino acid sequenceactually encoded by the sequenced DNA molecule, beginning at the pointof such an insertion or deletion.

[0059] By “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U).

[0060] Using the information provided herein, such as the nucleotidesequence in FIGS. 1A and B (SEQ ID NO:1), or the nucleotide sequence inFIGS. 20A and B (SEQ ID NO:19) a nucleic acid molecule (i.e.,polynucleotide) of the present invention encoding a TNF-gamma-alpha orTNF-gamma-beta polypeptide may be obtained using standard cloning andscreening procedures, such as, for example, those for cloning cDNAsusing mRNA as the starting material. For example, polynucleotidesencoding TNF-gamma-alpha polypeptides may routinely be obtained from anycell or tissue source that expresses TNF-gamma-alpha, such as, forexample, human kidney and umbilical vein endothelial cells. Illustrativeof the invention, the nucleic acid molecule described in FIGS. 1A and B(SEQ ID NO:1) was discovered in a cDNA library derived from humanumbilical vein endothelial cells. The cDNA clone corresponding toTNF-gamma-alpha (clone HUVE091) contains an open reading frame encodinga protein of 174 amino acid residues of which approximately the first 27amino acids residues are the putative leader sequence such that themature protein comprises 147 amino acids. The protein exhibits thehighest degree of homology at the C-terminus to Rabbit TNF-alpha (Ito,H., et al., DNA 5:157-165 (1986); GenBank Accession No. M12846; SEQ IDNO:7) with 38% identity and 58% similarity over a 111 amino acidstretch. Sequences conserved throughout the members of the TNF familyare also conserved in TNF-gamma (see FIGS. 2A-2C). The shaded lettersindicate conserved amino acid residues. The TNF-gamma mRNA isspecifically expressed in human umbilical vein endothelial cells asshown in the RNA blot analysis of FIG. 3B.

[0061] Further, polynucleotides encoding a TNF-gamma-beta polypeptidemay routinely be obtained from induced and resting endothelial cells,umbilical vein, tonsils, and several other cell and tissue types.Illustrative of the invention, the nucleic acid molecules described inFIGS. 20A and B (SEQ ID NO:19) was discovered in a cDNA library derivedfrom induced endothelial cells. The cDNA clone corresponding toTNF-gamma-beta (HEMCZ56) contains an open reading frame encoding aprotein of 251 amino acid residues of which approximately the first 35amino acid residues are the putative intracellular domain and aminoacids 36-61 are a putative transmembrane domain and amino acid residues62-251 are a putative extracellular domain. In specific embodiments, themature form of TNF-gamma-beta is amino acid residues 72-251 of theprotein encoded by the cDNA clone HEMCZ56.

[0062] In another embodiment, the cDNA clone corresponding toTNF-gamma-beta (HEMCZ56) contains an open reading frame encoding aprotein of 251 amino acid residues of which approximately the first 35amino acid residues are the putative intracellular domain and aminoacids 36-59 are a putative transmembrane domain and amino acid residues60-251 are a putative extracellular domain.

[0063] In one embodiment, the polynucleotides of the invention comprise,or alternatively consist of, the sequence shown in SEQ ID NO:25. Apolynucleotide comprising the nucleotide sequence provided as SEQ IDNO:25 was constructed by PCR amplification of the TNF-gamma-betapolynucleotide shown as SEQ ID NO:19 in a two-step process. The firstPCR reaction used the following primer pair.

[0064] Nde 1-169 (Nde I site with 1-169bp):          5′-GGA ATT CCA TATGCT GAA AGG TCA AGA ATT GCG ACC (SEQ ID NO: 27) GTC CCA CCA GCA GGT TTACG CAC CGC TGC GTG CAG ACG GTG ATA AGC CGC GTG CAC ACC TGA CCG TTG TGCGCC AGA CCC CGC CCC AGC ACT TCA AAA ACC AGT TCC CGG CTC TGC ACT GGG AGCACG AAC TGG GCC TGG CCT TCA-3′

[0065] 151-329 BstXI (151-329bp which contains BstXI site at 3′):         5′-ATC ACC ACG GTG ATG GAG TCC GGC TTG TTC GGA CGG (SEQ ID NO:28) CCT GCC TGA CGG ATT TCG GAG CAC TCA GAG GTC ATA CCA CGG AAG GTC ACCTGG GAG TAG ATG AAG TAG TCA CCA GAC TCC GGG ATC AGC AGG AAT TTG TTG GTGTAG TTC ATG CGG TTC TTG GTG AAG GCC AGG CCC AGT TC-3′

[0066] A second PCR reaction used the following primer pair:

[0067] BstXI 311-441 (311-441bp which contains BstXI site at 5′):         5′-ACT CCA TCA CCG TGG TGA TCA CCA AAG TGA CCG ACT (SEQ ID NO:29) CTT ACC CGG AGC CGA CCC AGC TGC TGA TGG GTA CCA AGT CTG TTT GCG AAGTTG GTT CCA ACT GGT TCC AGC CGA TCT ACC TCG GTG CCA TGT TC-3′

[0068] 521-546 Xba (521-546bp+Xba site):           5′-GCG TCT AGA TTATTA CAG CAG GAA GGC ACC GAA GAA (SEQ ID NO: 30) GGT TTT ATC TTC CTT GGTGTA ATC CAC CAG AGA GAT GTC GGA CAC GIT CAC CAT CAG TTT GTC GCC CTC TTGCAG GGA GAA CAT GGC ACC GAG GTA GAT-3′

[0069] A codon-optimized form of TNF-gamma-beta resulted fromrestricting the PCR products with Nde I and BstXI (first pair), BstXIand Xba (second pair), and then ligating the restricted productstogether into precut pHE4b vector (Nde and Xba). The amino acid sequenceresulting from the translation of SEQ ID NO:25 is provided as SEQ IDNO:26. Fragments, variants, and derivatives of the sequences provided asSEQ ID NO:25 and SEQ ID NO:26 are also encompassed by the invention. Incertain embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotideof SEQ ID NO:25. The present invention also encompasses the abovepolynucleotide sequences fused to a heterologous polynucleotidesequences as described herein and as are well known in the art.Polypeptides encoded by these nucleic acids and/or polynucleotidesequences are also encompassed by the invention.

[0070] The amino acid residues constituting the extracellular,transmembrane, and intracellular domains have been predicted by computeranalysis. Thus, as one of ordinary skill would appreciate, the aminoacid residues constituting these domains may vary slightly (e.g., byabout 1 to about 15 amino acid residues) depending on the criteria usedto define each domain.

[0071] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of FIGS. 1Aand B (SEQ ID NO:2), or for the mature polypeptide encoded by the cDNAof the clone designated RUVEO91 deposited as ATCC Deposit No. 75927 onOct. 26, 1994.

[0072] In addition, in accordance with another aspect of the presentinvention, there is provided an isolated nucleic acid (polynucleotide)which encodes for the mature polypeptide having the deduced amino acidsequence of FIGS. 20A and B (SEQ ID NO:20), or for the maturepolypeptide encoded by the cDNA of the clone designated HEMCZ56deposited as ATCC Deposit No. 203055 on Jul. 9, 1998.

[0073] By “isolated” nucleic acid molecule(s) or polynucleotide isintended a molecule, DNA or RNA, which has been removed form its nativeenvironment. For example, recombinant DNA molecules (polynucleotides)contained in a vector are considered isolated for the purposes of thepresent invention. Further examples of isolated DNA molecules(polynucleotides) include recombinant DNA molecules maintained inheterologous host cells or purified (partially or substantially) DNAmolecules in solution. Isolated RNA molecules (polynucleotides) includein vivo or in vitro RNA transcripts of the DNA molecules(polynucleotides) of the present invention. However, a nucleic acidcontained in a clone that is a member of a library (e.g., a genomic orcDNA library) that has not been isolated from other members of thelibrary (e.g., in the form of a homogeneous solution containing theclone and other members of the library) or a chromosome isolated orremoved from a cell or a cell lysate (e.g., a “chromosome spread”, as ina karyotype), or a genomic DNA preparation (either intact, ormechanically and/or enzymatically sheared) is not “isolated” for thepurposes of this invention. Isolated nucleic acid molecules orpolynucleotides according to the present invention further include suchmolecules produced synthetically.

[0074] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand.

[0075] Isolated nucleic acid molecules of the present invention includethe polynucleotide sequence depicted in FIGS. 1A and B (SEQ ID NO:1)encoding the full-length and/or mature TNF-gamma-alpha polypeptide, thepolynucleotide sequence depicted in FIGS. 20A and B (SEQ ID NO:19)encoding the full-length and/or mature TNF-gamma-beta polypeptide, thepolynucleotide sequences contained in deposited clone (HUVE091)deposited as ATCC Deposit No. 75927 encoding the full-length and/ormature TNF-gamma-alpha polypeptide, the polynucleotide sequencescontained in deposited clone (HEMCZ56) deposited as ATCC Deposit No.203055 encoding the full-length and/or mature TNF-gamma-betapolypeptide, and polynucleotide sequences which comprise a sequencedifferent from those described above, but which due to the degeneracy ofthe genetic code, encode the same full-length and/or mature polypeptideas the DNA of FIGS. 1A and B, FIGS. 20A and B, or the deposited cDNAs.Of course, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate such degenerate variants.

[0076] The amino acid sequence of the complete TNF-gamma-alpha proteinincludes a leader sequence and a mature protein, as shown in FIGS. 1Aand B (SEQ ID NO:2). The amino acid sequence of the completeTNF-gamma-beta protein includes a leader sequence and a mature protein,as shown in FIGS. 20A and B (SEQ ID NO:20). More in particular, thepresent invention provides nucleic acid molecules encoding a mature formof the TNF-gamma-alpha protein. The present invention also providesnucleic acid molecules encoding a mature form of the TNF-gamma-betaprotein. Thus, according to the signal hypothesis, once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the complete polypeptideto produce a secreted “mature” form of the protein. Most mammalian cellsand even insect cells cleave secreted proteins with the samespecificity. However, in some cases, cleavage of a secreted protein isnot entirely uniform, which results in two or more mature species of theprotein. Further, it has long been known that the cleavage specificityof a secreted protein is ultimately determined by the primary structureof the complete protein, that is, it is inherent in the amino acidsequence of the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature TNF-gamma-alpha polypeptidehaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No. 75927. The present invention also provides a nucleotidesequence encoding the mature TNF-gamma-beta polypeptide having the aminoacid sequence encoded by the cDNA clone contained in ATCC Deposit No.203055. By the “mature TNF-gamma-alpha polypeptide having the amino acidsequence encoded by the cDNA clone in ATCC Deposit No. 75927” is meantthe mature form(s) of the TNF-gamma-alpha protein produced by expressionin a mammalian cell (e.g., COS cells, as described below) of thecomplete open reading frame encoded by the human DNA sequence of thedeposited clone. Likewise, by the “mature TNF-gamma-beta polypeptidehaving the amino acid sequence encoded by the cDNA clone in ATCC DepositNo. 203055” is meant the mature form(s) of the TNF-gamma-beta proteinproduced by expression in a mammalian cell (e.g., COS cells, asdescribed below) of the complete open reading frame encoded by the humanDNA sequence of the deposited clone.

[0077] The polynucleotide which encodes for the mature polypeptide ofFIGS. 20A and B or for the mature polypeptide encoded by the depositedcDNA (HEMCZ56) may include, but is not limited to: only the codingsequence for the mature polypeptide; the coding sequence for the maturepolypeptide and additional coding sequence such as a leader or secretorysequence or a transmembrane sequence or a proprotein sequence; thecoding sequence for an extracellular domain; the coding sequence for themature polypeptide (and optionally additional coding sequence) andnon-coding sequence, such as introns or non-coding sequence 5′ and/or 3′of the coding sequence for the mature polypeptide.

[0078] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0079] Thus, for example, the polynucleotide of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and a presequence (leadersequence).

[0080] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0081] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0082] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments (i.e.,portions), analogs and derivatives of the polypeptide having the deducedamino acid sequence of FIGS. 1A and B, FIGS. 20A and B, and thepolypeptide encoded by the cDNA of the deposited clones. The variant ofthe polynucleotide may be a naturally occurring allelic variant of thepolynucleotide or a non-naturally occurring variant of thepolynucleotide.

[0083] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIGS. 1A and B, or the maturepolypeptide encoded by the cDNA of the deposited clone HUVEO91 as wellas variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIGS. 1A and B, orthe polypeptide encoded by the cDNA of the deposited clone HUVEO91. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

[0084] Additionally, the present invention includes polynucleotidesencoding the mature polypeptide as shown in FIGS. 20A and B, asdescribed herein, or the mature polypeptide encoded by the cDNA of thedeposited clone HEMCZ56 as well as variants of such polynucleotideswhich variants encode for a fragment, derivative or analog of thepolypeptide of FIGS. 20A and B, the polypeptide as described herein, orthe polypeptide encoded by the cDNA of the deposited clone HEMCZ56. Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

[0085] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1A and 1B or of the coding sequence of thedeposited clone HUVEO91. Alternatively, the polynucleotide may have acoding sequence which is a naturally occurring allelic variant of thecoding sequence shown in FIGS. 20A and B or of the coding sequence ofthe deposited clone HEMCZ56. As known in the art, an allelic variant isan alternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

[0086] The present invention is further directed to fragments of theisolated nucleic acid molecules described herein. By a fragment of anisolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNAs (HUVEO9 1 and HEMCZ56), or the nucleotide sequence shownin FIGS. 1A and B (SEQ ID NO:1), FIGS. 20A and B (SEQ ID NO:19), or thecomplementary strand thereto, is intended fragments at least 15 nt, andmore preferably at least 20 nt, still more preferably at least 30 nt,and even more preferably, at least 40, 50, 100, 150, 200, 250, 300, 400,or 500 nt in length. These fragments have numerous uses which include,but are not limited to, diagnostic probes and primers as discussedherein. Of course, larger fragments 50-1500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequence of the deposited cDNA cloneHUVEO91, the deposited cDNA clone HEMCZ56, the nucleotide sequencedepicted in FIGS. 1A and B (SEQ ID NO:1), or the nucletoide sequencedepicted in FIGS. 20A and B (SEQ ID NO 20). By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNAclones (HUVEO91 and HEMCZ56), the nucleotide sequence as shown in FIGS.1A and B (SEQ ID NO:1), or the nucleotide sequence as shown in FIGS. 20Aand B.

[0087] In specific embodiments, the polynucleotide fragments of theinvention encode a polypeptide which demonstrates a functional activity.By a polypeptide demonstrating “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a complete or mature TNF-gamma polypeptide.Such functional activities include, but are not limited to, biologicalactivity ((e.g., inhibition of angiogenesis, inhibition of endothelialcell proliferation, induction of NF-kappaB and c-Jun kinase (JNK),induction of cell adhesion, and induction of apoptosis (See Examples,particularly Examples 12-15) induction of T cell proliferation andsecretion of interferon-gamma and/or GM-CSF by T cells, exacerbation ofan in-vivo mixed-lymphocyte reaction (see Examples 35-37), antigenicity[ability to bind (or compete with a TNF-gamma polypeptide for binding)to an anti-TNF-gamma antibody], immunogenicity (ability to generateantibody which binds to a TNF-gamma polypeptide), the ability to formpolymers with other TNF-gamma polypeptides, and ability to bind to areceptor or ligand for a TNF-gamma polypeptide (e.g. DR3 (InternationalPublication Numbers WO97/33904 and WO00/64465 and TR6 (InternationalPublication Numbers WO98/30694 and WO00/52028).

[0088] The invention also provides nucleic acid molecules havingnucleotide sequences related to extensive fragments of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HUVEO91(SEQ ID NO:8), HMPAP05 (SEQ ID NO:9), HSXCA44 (SEQ ID NO:10), HEMFG66(SEQ ID NO:11), and HELAM93 (SEQ ID NO:12).

[0089] The invention also provides nucleic acid molecules havingnucleotide sequences related to extensive fragments of SEQ ID NO:19which have been determined from the following related cDNA clones:HUVEO91P01 (SEQ ID NO:21), HMPTI24R (SEQ ID NO:22), HELAM93R (SEQ IDNO:23), and HEMFG66R (SEQ ID NO:24).

[0090] In specific embodiments, the polynucleotide fragments of theinvention comprise, or alternatively, consist of, a polynucleotidecomprising any portion of at least 30 nucleotides, preferably at least50 nucleotides, of SEQ ID NO:1 from nucleotide residue 1 to 2442,preferably excluding the nucleotide sequences determined from the abovelisted cDNA clones. Representative examples of the TNF-gamma-alphapolynucleotide fragments of the invention, include fragments thatcomprise, or alternatively, consist of, a member selected from the groupconsisting of nucleotides: 783-1304, 800-1300, 850-1300, 900-1300,950-1300, 1000-1300, 1050-1300, 1100-1300, 1150-1300, 1200-1300,1250-1300, 783-1250, 800-1250, 850-1250, 900-1250, 950-1250, 1000-1250,1050-1250, 1100-1250, 1150-1250, 1200-1250, 783-1200, 800-1200,850-1200, 900-1200, 950-1200, 1000-1200, 1050-1200, 1100-1200,1150-1200, 783-1150, 800-1150, 850-1150, 900-1150, 950-1150, 1000-1150,1050-1150, 1100-1150, 783-1100, 800-1100, 850-1100, 900-1100, 950-1100,1000-1100, 1050-1100, 783-1050, 800-1050, 850-1050, 900-1050, 950-1050,1000-1050, 783-1000, 800-1000, 850-1000, 900-1000, 950-1000, 783-950,800-950, 850-950, 900-950, 783-900, 800-900, and 850-900 of SEQ ID NO:1,or the complementary polynucleotide strand thereto, or the cDNAcontained in the deposited clone HUVEO91. Polypeptides encoded by thesepolynucleotide fragments are also encompassed by the invention. Incertain embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotidesequence described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequences as described herein and as are well known in the art.Polypeptides encoded by these nucleic acids and/or polynucleotidesequences are also encompassed by the invention.

[0091] In additional specific embodiments, the polynucleotide fragmentsof the invention comprise, or alternatively, consist of, apolynucleotide comprising any portion of at least 30 nucleotides,preferably at least 50 nucleotides, of SEQ ID NO:19 from nucleotideresidue 1 to 1116, preferably excluding the nucleotide sequencesdetermined from the above listed cDNA clones (i.e., list from p.25).

[0092] Preferred embodiments of the invention encompass polynucleotidesencoding polypeptides comprising, or alternatively consisting of, amember selected from the group consisting of the amino acid sequence ofresidues −1-147 (i.e., −1 to 147), 1-147 (i.e., +1 to 147), 2-147,3-147, 4-147, 5-147, 6-147, 7-147, 8-147, 9-147, 10-147, 11-147, 12-147,and 13-147 of SEQ ID NO:2. Polynucleotides encoding these polypeptidesare also provided.

[0093] Representative examples of the TNF-gamma-beta polynucleotidefragments of the invention, include fragments that comprise, oralternatively, consist of, a member selected from the group consistingof nucleotides 1-1116, 50-1116, 100-1116, 150-1116, 200-1116, 250-1116,300-1116, 350-1116, 400-1116, 450-1116, 500-1116, 550-1116, 600-1116,650-1116, 700-1116, 750-1116, 800-1116, 850-1116, 900-1116, 950-1116,1000-1116, 1050-1116, 1-1100, 50-1100, 100-1100, 150-1100, 200-1100,250-1100, 300-1100, 350-1100, 400-1100, 450-1100, 500-1100, 550-1100,600-1100, 650-1100, 700-1100, 750-1100, 800-1100, 850-1100, 900-1100,950-1100, 1000-1100, 1050-1100, 1-1050, 50-1050, 100-1050, 150-1050,200-1050, 250-1050, 300-1050, 350-1050, 400-1050, 450-1050, 500-1050,550-1050, 600-1050, 650-1050, 700-1050, 750-1050, 800-1050, 850-1050,900-1050, 950-1050, 1000-1050, 1-1000, 50-1000, 100-1000, 150-1000,200-1000, 250-1000, 300-1000, 350-1000, 400-1000, 450-1000, 500-1000,550-1000, 600-1000, 650-1000, 700-1000, 750-1000, 800-1000, 850-1000,900-1000, 950-1000, 1-950, 50-950, 100-950, 150-950, 200-950, 250-950,300-950, 350-950, 400-950, 450-950, 500-950, 550-950, 600-950, 650-950,700-950, 750-950, 800-950, 850-950, 900-950, 1-900, 50-900, 100-900,150-900, 200-900, 250-900, 300-900, 350-900, 400-900, 450-900, 500-900,550-900, 600-900, 650-900, 700-900, 750-900, 800-900, 850-900, 1-850,50-850, 100-850, 150-850, 200-850, 250-850, 300-850, 350-850, 400-850,450-850, 500-850, 550-850, 600-850, 650-850, 700-850, 750-850, 800-850,1-800, 50-800, 100-800, 150-800, 200-800, 250-800, 300-800, 350-800,400-800, 450-800, 500-800, 550-800, 600-800, 650-800, 700-800, 750-800,1-750, 50-750, 100-750, 150-750, 200-750, 250-750, 300-750, 350-750,400-750, 450-750, 500-750, 550-750, 600-750, 650-750, 700-750, 1-700,50-700, 100-700, 150-700, 200-700, 250-700, 300-700, 350-700, 400-700,450-700, 500-700, 550-700, 600-700, 650-700, 1-650, 50-650, 100-650,150-650, 200-650, 250-650, 300-650, 350-650, 400-650, 450-650, 500-650,550-650, 600-650, 1-600, 50-600, 100-600, 150-600, 200-600, 250-600,300-600, 350-600, 400-600, 450-600, 500-600, 550-600, 1-550, 50-550,100-550, 150-550, 200-550, 250-550, 300-550, 350-550, 400-550, 450-550,500-550, 1-500, 50-500, 100-500, 150-500, 200-500, 250-500, 300-500,350-500, 400-500, 450-500, 1-450, 50-450, 100-450, 150-450, 200-450,250-450, 300-450, 350-450, 400-450, 1-400, 50-400, 100-400, 150-400,200-400, 250-400, 300-400, 350-400, 1-350, 50-350, 100-350, 150-350,200-350, 250-350, 300-350, 1-300, 50-300, 100-300, 150-300, 200-300,250-300, 1-250, 50-250, 100-250, 150-250, 200-250, 1-200, 50-200,100-200, 150-200, 1-150, 50-150, 100-150, 1-100, 50-100, and 1-50 of SEQNO:19 or the complementary polynucleotide strand thereto, or the cDNAcontained in the deposited clone HEMCZ56. Polypeptides encoded by thesepolynucleotide fragments are also encompassed by the invention. Incertain embodiments, polynucleotides of the invention comprise, oralternatively consist of, a polynucleotide sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotidesequence described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequences as described herein and as are well known in the art.Polypeptides encoded by these nucleic acids and/or polynucleotidesequences are also encompassed by the invention.

[0094] Preferred nucleic acid fragments of the present invention alsoinclude nucleic acid molecules encoding one or more of the followingdomains of TNF-gamma-alpha (e.g., as described also in the legend toFIGS. 1A and 1B): potential asparagine-linked glycosylation sites N-29through N-32 (N-29, Y-30, T-31, N-32) and N-125 through D-128 (N-125,V-126, S-127, D-128); potential Protein Kinase C (PKC) phosphorylationsites T-32 through K-34 (T-32, N-33, K-34) and T-50 through R-52 (T-50,F-51, R-52); potential Casein Kinase II (CK2) phosphorylation sites S-83through E-86 (S-83, Y-84, P-85, E-86); S-96 through E-99 (S-96, V-97,C-98, E-99); S-115 through E-118 (S-115, L-116, Q-117, E-118); S-130through D-133 (S-130, L-131, V-132, D-133); and T-135 through D-138(T-135, K-136, E-137, D-138); and potential myristylation sites G-20through K-25 (G-20, L-21, A-22, F-23, T-24, K-25) and G-111 throughL-116 (G-111, A-112, M-113, F-114, S-115, L-116) of SEQ ID NO:2.

[0095] Among the especially preferred polynucleotides of the inventionare those characterized by encoding structural or functional attributesof TNF-gamma. Such polynucleotides encode amino acid residues thatcomprise alpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet forming regions (“beta regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., having an antigenic regions ofthree or more contiguous amino acid residues each of which having anantigenic index of greater than or equal to 1.5) of TNF-gamma. Certainpreferred regions are those set out in FIG. 17, and include, but are notlimited to, regions of the aforementioned types identified by analysisof the amino acid sequence depicted in FIG. 1 (SEQ ID NO:2) using thedefault parameters of the identified computer programs, such preferredregions include; Garnier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions,and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobicregions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf regionsof high antigenic index.

[0096] Data which represent TNF-gamma-beta in a fashion as describedabove for TNF-gamma-alpha (see FIG. 17) may easily be prepared using theamino acid sequence shown in FIGS. 20A and 20B and in SEQ ID NO:20. Assuch, each of the above listed structural or functional attributes ofTNF-gamma listed above (i.e. Garnier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions,and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobicregions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulzflexible regions, Emini surface-forming regions and Jameson-Wolf regionsof high antigenic index, etc.) apply equally well to TNF-gamma-alpha andTNF-gamma-beta.

[0097] Certain preferred regions in these regards are set out in FIG.17, but may also be represented or identified by using a tabularrepresentation of the data presented in FIG. 17. The DNA*STAR computeralgorithm used to generate FIG. 17 (set on the original defaultparameters) will easily present the data in FIG. 17 in such a tabularformat. A tabular format of the data in FIG. 17 may be used to easilydetermine specific boundaries of a preferred region.

[0098] The above-mentioned preferred regions set out in FIG. 17 include,but are not limited to, regions of the aforementioned types identifiedby analysis of the amino acid sequence set out in FIGS. 1A and 1B. Asset out in FIG. 17, such preferred regions include Garnier-Robsonalpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasmanalpha-regions, beta-regions, and coil-regions, Kyte-Doolittlehydrophilic regions and hydrophobic regions, Eisenberg alpha- andbeta-amphipathic regions, Karplus-Schulz flexible regions, Eminisurface-forming regions and Jameson-Wolf regions of high antigenicindex.

[0099] Among highly preferred fragments in this regard are those thatcomprise regions of TNF-gamma-alpha and/or TNF-gamma-beta that combineseveral structural features, such as several (e.g., 1, 2, 3 or 4) of thefeatures set out above.

[0100] Additional preferred nucleic acid fragments of the presentinvention include nucleic acid molecules encoding one or moreepitope-bearing portions of the TNF-gamma polypeptide. In particular,such nucleic acid fragments of the present invention include nucleicacid molecules encoding a member selected from the group consisting of:a polypeptide comprising amino acid residues from about Thr-24 to aboutAsn-32 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Ile-37 to about Ile-45 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Met-54 to about Arg-62 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Gln-63 to aboutAsp-71 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Glu-57 to about Gly-65 in SEQ ID NO:2; a polypeptide comprisingamino acid residues from about Val-80 to about Thr-88 in SEQ ID NO:2; apolypeptide comprising amino acid residues from about Leu-116 to aboutVal-124 in SEQ ID NO:2; and a polypeptide comprising amino acid residuesfrom about Asp-133 to about Phe-141 in SEQ ID NO:2. These polypeptidefragments have been determined to bear antigenic epitopes of theTNF-gamma protein by the analysis of the Jameson-Wolf antigenic index,as shown in FIG. 17, above. Methods for determining other suchepitope-bearing portions of TNF-gamma are described in detail below.

[0101] Polypeptide fragments which bear antigenic epitopes of theTNF-gamma-beta protein may be easily determined by one of skill in theart using the above-described analysis of the Jameson-Wolf antigenicindex, as shown in FIG. 17. Methods for determining other suchepitope-bearing portions of TNF-gamma-beta are described in detailbelow.

[0102] Another embodiment of the invention is directed topolynucleotides that hybridize, preferably under stringent hybridizationconditions, to a portion of the polynucleotide sequence of apolynucleotide of the invention such as, for instance, the cDNA clonecontained in ATCC Deposit No. 75927, the cDNA clone contained in ATCCDeposit 203055 or a TNF-gamma polynucleotide fragment as describedherein. By “stringent hybridization conditions” is intended overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (750mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 micrograms/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C. By a polynucleotide which hybridizes to a“portion” of a polynucleotide is intended a polynucleotide (either DNAor RNA) hybridizing to at least 15 nucleotides (nt), and more preferablyat least 20 nt, still more preferably at least 30 nt, and even morepreferably 30-70, or 80-150 nt, or the entire length of the referencepolynucleotide. These are useful as diagnostic probes and primers asdiscussed above and in more detail below. Of course, a polynucleotidewhich hybridizes only to a poly A sequence (such as the 3′ terminal polytract of the TNF-gamma cDNA shown in SEQ ID NO:1 or SEQ ID NO:19), or toa complementary stretch of T (or U) residues, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNA clonegenerated using oligo dT as a primer).

[0103] In preferred embodiments, polynucleotides which hybridize to thereference polynucleotides disclosed herein encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the polynucleotide sequences depictedin FIGS. 1A and 1B (SEQ ID NO:1) and/or FIGS. 20A and B (SEQ ID NO:19),or the cDNAs contained in the deposit.

[0104] Alternative embodiments are directed to polynucleotides whichhybridize to the reference polynucleotide (i.e., a polynucleotidesequence disclosed herein), but do not retain biological activity. Whilethese polynucleotides do not retain biological activity, they have uses,such as, for example, as probes for the polynucleotide of SEQ ID NO:1,for recovery of the polynucleotide, as diagnostic probes, and as PCRprimers.

[0105] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode portions, analogsor derivatives of the TNF-gamma protein. Variants may occur naturally,such as a natural allelic variant. By an “allelic variant” is intendedone of several alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

[0106] Such variants include those produced by nucleotide substitutions,deletions or additions of the polynucleotide sequences described herein(including fragments). The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the TNF-gamma protein or portions thereof.Also especially preferred in this regard are conservative substitutions.

[0107] Further embodiments of the invention are directed to isolatednucleic acid molecules comprising a polynucleotide sequence at least 70%or at least 80% or 85% identical, more preferably at least 90%, 92% or94% identical, and still more preferably at least 95%, 96%, 97%, 98% or99% identical to a polynucleotide having a nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence encoding theTNF-gamma-alpha polypeptide having the complete amino acid sequence inSEQ ID NO:2 (i.e., positions −27 to 147 of SEQ ID NO:2); (b) anucleotide sequence encoding the TNF-gamma-alpha polypeptide having thecomplete amino acid sequence in SEQ ID NO:2 excepting the N-terminalmethionine (i.e., positions −26 to 147 of SEQ ID NO:2); (c) a nucleotidesequence encoding the mature TNF-gamma-alpha polypeptide having theamino acid sequence in SEQ ID NO:2 shown as positions 1 to 147 of SEQ IDNO:2; (d) a nucleotide sequence encoding the extracellular domain of theTNF-gamma-alpha polypeptide having the amino acid sequence in SEQ IDNO:2 shown as positions 1 to 147 of SEQ ID NO:2; (e) a nucleotidesequence encoding the TNF-gamma-alpha polypeptide having the completeamino acid sequence encoded by the cDNA clone HUVEO91 contained in ATCCDeposit No. 75927; (f) a nucleotide sequence encoding theTNF-gamma-alpha polypeptide having the complete amino acid sequenceexcepting the N-terminal methionine encoded by the cDNA clone HUVEO91contained in ATCC Deposit No. 75927; (g) a nucleotide sequence encodingthe mature TNF-gamma-alpha polypeptide having the amino acid sequenceencoded by the cDNA clone HUVEO91 contained in ATCC Deposit No. 75927;(h) a nucleotide sequence encoding the extracellular domain of theTNF-gamma-alpha polypeptide having the amino acid sequence encoded bythe cDNA clone HUVEO91 contained in ATCC Deposit No. 75927; (i) anucleotide sequence encoding a polypeptide fragment described herein;and (j) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), (h) or (i), above. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% or 85% identical, more preferably atleast 90%, 92% or 94% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e),(f), (g), (h), (i) or (j), above, as well as polypeptides having anamino acid sequence with at least 90% similarity, and more preferably atleast 95% similarity, to those above.

[0108] Further embodiments of the invention are directed to isolatednucleic acid molecules comprising a polynucleotide sequence at least 70%or at least 80% or 85% identical, more preferably at least 90%, 92% or94% identical, and still more preferably at least 95%, 96%, 97%, 98% or99% identical to a polynucleotide having a nucleotide sequence selectedfrom the group consisting of: (a) a nucleotide sequence encoding theTNF-gamma-beta polypeptide having the complete amino acid sequence inSEQ ID NO:20 (i.e., positions 1 to 251 of SEQ ID NO:20); (b) anucleotide sequence encoding the TNF-gamma-beta polypeptide having thecomplete amino acid sequence in SEQ ID NO:20 excepting the N-terminalmethionine (i.e., positions 2 to 251 of SEQ ID NO:20); (c) a nucleotidesequence encoding the mature TNF-gamma-beta polypeptide having the aminoacid sequence in SEQ ID NO:20 shown as positions 62 to 251 of SEQ IDNO:20; (d) a nucleotide sequence encoding the mature TNF-gamma-betapolypeptide having the amino acid sequence in SEQ ID NO:20 shown aspositions 60 to 251 of SEQ ID NO:20; (e) a nucleotide sequence encodinga mature TNF-gamma-beta polypeptide having the amino acid sequence inSEQ ID NO:20 shown as positions 72 to 251 of SEQ ID NO:20; (f) anucleotide sequence encoding the intracellular domain of theTNF-gamma-beta polypeptide having the amino acid sequence in SEQ IDNO:20 shown as positions 1 to 35 of SEQ ID NO:20; (g) a nucleotidesequence encoding the intracellular domain of the TNF-gamma-betapolypeptide having the amino acid sequence in SEQ ID NO:20 shown aspositions 1 to 35 of SEQ ID NO:20; (h) a nucleotide sequence encodingthe extracellular domain of the TNF-gamma-beta polypeptide having theamino acid sequence in SEQ ID NO:20 shown as positions 62 to 251 of SEQID NO:20; (i) a nucleotide sequence encoding the extracellular domain ofthe TNF-gamma-beta polypeptide having the amino acid sequence in SEQ IDNO:20 shown as positions 60 to 251 of SEQ ID NO:20; (j) a nucleotidesequence encoding the extracellular domain of the TNF-gamma-betapolypeptide having the amino acid sequence in SEQ ID NO:20 shown aspositions 62 to 251 of SEQ ID NO:20; (k) a nucleotide sequence encodingthe TNF-gamma-beta polypeptide having the complete amino acid sequenceencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;(l) a nucleotide sequence encoding the TNF-gamma-beta polypeptide havingthe complete amino acid sequence excepting the N-terminal methionineencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;(m) a nucleotide sequence encoding the mature TNF-gamma-beta polypeptidehaving the amino acid sequence encoded by the cDNA clone HEMCZ56contained in ATCC Deposit No. 203055; (n) a nucleotide sequence encodingthe intracellular domain of the TNF-gamma-beta polypeptide having theamino acid sequence encoded by the cDNA clone HEMCZ56 contained in ATCCDeposit No. 203055; (o) a nucleotide sequence encoding the extracellulardomain of the TNF-gamma-beta polypeptide having the amino acid sequenceencoded by the cDNA clone HEMCZ56 contained in ATCC Deposit No. 203055;and (p) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l),(m), (n), (o) or (p), above. The polypeptides of the present inventionalso include polypeptides having an amino acid sequence at least 80% or85% identical, more preferably at least 90%, 92% or 94% identical, andstill more preferably 95%, 96%, 97%, 98% or 99% identical to thosedescribed in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l),(m), (n) or (o), above, as well as polypeptides having an amino acidsequence with at least 90% similarity, and more preferably at least 95%similarity, to those above.

[0109] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence of thepresent invention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding theTNF-gamma polypeptide. In other words, to obtain a polynucleotide havinga nucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The reference (query)sequence may be the entire nucleotide sequence shown in FIGS. 1A and B(SEQ ID NO:1) and FIGS. 20A and B (SEQ ID NO:19), or any fragment asdescribed herein.

[0110] As a practical matter, whether any particular nucleic acidmolecule is at least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or99% identical to, for instance, the nucleotide sequence shown in FIGS.1A and B (SEQ ID NO:1), FIGS. 20A and B (SEQ ID NO:19), or to thenucleotide sequence of the deposited cDNA clones can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0111] In a specific embodiment, the identity between a reference(query) sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB alignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

[0112] In further embodiments, the present invention is directed topolynucleotides having at least a 70% identity, preferably at least 80%,85% or 90% and more preferably at least a 92%, 94%, 95%, 96%, 97%, 98%or 99% identity to a polynucleotide which encodes the polypeptide of SEQID NO:2 as well as fragments thereof, which fragments have at least 30bases and preferably at least 50 bases and to polypeptides encoded bysuch polynucleotides.

[0113] In further embodiments, the present invention is directed topolynucleotides having at least a 70% identity, preferably at least 90%and more preferably at least a 95% identity to a polynucleotide whichencodes the polypeptide of SEQ ID NO:20 as well as fragments thereof,which fragments have at least 30 bases and preferably at least 50 basesand to polypeptides encoded by such polynucleotides.

[0114] The present application is directed to nucleic acid molecules atleast 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the polynucleotide sequence shown in FIGS. 1A and B (SEQ ID NO:1),FIGS. 20A and B (SEQ ID NO:19), or to the nucleic acid sequence of thedeposited cDNA clones, or fragments thereof, irrespective of whetherthey encode a polypeptide having TNF-gamma functional activity. This isbecause even where a particular nucleic acid molecule does not encode apolypeptide having TNF-gamma functional activity, one of skill in theart would still know how to use the nucleic acid molecule, for instance,as a hybridization probe or a polymerase chain reaction (PCR) primer.Uses of the nucleic acid molecules of the present invention that do notencode a polypeptide having TNF-gamma functional activity include, interalia, (1) isolating the TNF-gamma gene or allelic variants thereof in acDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theTNF-gamma gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, N.Y. (1988); and (3)Northern Blot analysis for detecting TNF-gamma mRNA expression inspecific tissues.

[0115] Preferred, however, are nucleic acid molecules having sequencesat least 70%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%identical to the nucleic acid sequence shown in FIGS. 1A and B (SEQ IDNO:1), FIGS. 20A and B (SEQ ID NO:19), or to the nucleic acid sequenceof the deposited cDNA clones, or fragments thereof, which do, in fact,encode a polypeptide having TNF-gamma functional activity. By “apolypeptide having TNF-gamma functional activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the TNF-gamma polypeptide of the invention (either thefull-length protein or, preferably, the mature protein), as measured ina particular immunoassay and/or biological assay. For example, TNF-gammaactivity can be measured using an apoptosis assay as described inExample 7, by determining the relative ability of TNF-gamma to inhibitthe FGF-2-induced formation of capillary-like tubular structureformation in cultures of ABAE cells as described in detail in Example 9or in a chorioallantoic membrane (CAM) angiogenesis assay as describedin Example 10, by its effect(s) on the activation of cellular NF-kappaBand c-Jun kinase (JNK) as described in Example 12, and in severaladditional ways described in the remaining Examples and in the art.

[0116] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 70%, 80%, 85%,90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acidsequence of the deposited cDNA or the nucleic acid sequence shown inFIGS. 1A and 1B (SEQ ID NO:1), FIGS. 20A and 20B (SEQ ID NO:19), orfragments thereof, will encode a polypeptide “having TNF-gammaactivity.” In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed assay. It will be further recognized in the art that, for suchnucleic acid molecules that are not degenerate variants, a reasonablenumber will also encode a polypeptide having TNF-gamma activity. This isbecause the skilled artisan is fully aware of amino acid substitutionsthat are either less likely or not likely to significantly effectprotein function (e.g., replacing one aliphatic amino acid with a secondaliphatic amino acid). For example, guidance concerning how to makephenotypically silent amino acid substitutions is provided in J. U.Bowie et al., “Deciphering the Message in Protein Sequences: Toleranceto Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein theauthors indicate that proteins are surprisingly tolerant of amino acidsubstitutions.

[0117] Additional embodiments of the invention are directed to isolatednucleic acid molecules comprising a polynucleotide which encodes theamino acid sequence of a TNF-gamma polypeptide (e.g., a TNF-gammapolypeptide fragment described herein) having an amino acid sequencewhich contains at least one conservative amino acid substitution, butnot more than 50 conservative amino acid substitutions, even morepreferably, not more than 40 conservative amino acid substitutions,still more preferably, not more than 30 conservative amino acidsubstitutions, and still even more preferably, not more than 20conservative amino acid substitutions, 10-20 conservative amino acidsubstitutions, 5-10 conservative amino acid substitutions, 1-5conservative amino acid substitutions, 3-5 conservative amino acidsubstitutions, or 1-3 conservative amino acid substitutions. Of course,in order of ever-increasing preference, it is highly preferable for apolynucleotide which encodes the amino acid sequence of a TNF-gammapolypeptide to have an amino acid sequence which contains not more than10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.

[0118] Additional embodiments of the invention are directed toexclusions of publicly available polynucleotide sequences. Manypolynucleotide sequences, such as EST sequences, are publicly availableand accessible through sequence databases. Some of these sequences arerelated to SEQ ID NO:1 and/or SEQ ID NO:19 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Thus, preferably excluded from the present invention are one or morepolynucleotides comprising a nucleotide sequence described by thegeneral formula of a¹-b¹, where a¹ is any integer between 1 to 2410 ofSEQ ID NO:1, b¹ is an integer of 15 to 2425, where both a¹ and b¹correspond to the positions of nucleotide residues shown in SEQ ID NO:1,and where b¹ is greater than or equal to a¹+14. Similarly, preferablyexcluded from the present invention are one or more polynucleotidescomprising a nucleotide sequence described by the general formula ofa²-b², where a² is any integer between 1 to 1101 of SEQ ID NO:19, b² isan integer of 15 to 1116, where both a² and b² correspond to thepositions of nucleotide residues shown in SEQ ID NO:19, and where b² isgreater than or equal to a²+14.

[0119] In specific embodiments, the polynucleotides of the invention areless than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb inlength. In a further embodiment, polynucleotides of the inventioncomprise at least 15 contiguous nucleotides of TNF-gamma codingsequence, but do not comprise all or a portion of any TNF-gamma intron.In another embodiment, the nucleic acid comprising TNF-gamma codingsequence does not contain coding sequences of a genomic flanking gene(i.e., 5′ or 3′ to the TNF-gamma gene in the genome).

[0120] In specific embodiments, the polynucleotides of the invention areless than 100,000 kb, 50,000 kb, 10,000 kb, 1,000 kb, 500 kb, 400 kb,350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb,50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb inlength.

[0121] In further embodiments, polynucleotides of the invention compriseat least 15, at least 30, at least 50, at least 100, or at least 250, atleast 500, or at least 1000 contiguous nucleotides of TNF-gamma-alpha orTNF-gamma-beta coding sequence, but consist of less than or equal to1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5′ or 3′coding nucleotide sequences set forth in SEQ ID NO:1 or SEQ ID NO:19,respectively. In further embodiments, polynucleotides of the inventioncomprise at least 15, at least 30, at least 50, at least 100, or atleast 250, at least 500, or at least 1000 contiguous nucleotides ofTNF-gamma-alpha or TNF-gamma-beta coding sequence, but do not compriseall or a portion of any TNF-gamma intron. In another embodiment, thenucleic acid comprising TNF-gamma-alpha or TNF-gamma-beta codingsequence does not contain coding sequences of a genomic flanking gene(i.e., 5′ or 3′ to the TNF-gamma gene in the genome). In otherembodiments, the polynucleotides of the invention do not contain thecoding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5,4, 3, 2, or 1 genomic flanking gene(s).

Polynucleotide Assays

[0122] The invention also encompasses the use of TNF-gammapolynucleotides to detect complementary polynucleotides, such as, forexample, as a diagnostic reagent for detecting diseases orsusceptibility to diseases related to the presence of mutatedTNF-gamma-alpha or TNF-gamma-beta. Such diseases are related to anunder-expression of TNF-gamma-alpha or TNF-gamma-beta, such as, forexample, abnormal cellular proliferation such as tumors and cancers.

[0123] Individuals carrying mutations in the human TNF-gamma gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding TNF-gamma-alpha orTNF-gamma-beta can be used to identify and analyze TNF-gamma mutations.For example, deletions and insertions can be detected by a change insize of the amplified product in comparison to the normal genotype.Point mutations can be identified by hybridizing amplified DNA toradiolabeled TNF-gamma RNA or alternatively, radiolabeled TNF-gammaantisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0124] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0125] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0126] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0127] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0128] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

Vectors and Host Cells

[0129] The present invention also relates to vectors which include theisolated polynucleotides of the present invention, host cells which aregenetically engineered with the recombinant vectors, or which areotherwise engineered to produce the polypeptides of the invention, andthe production of polypeptides of the invention by recombinanttechniques.

[0130] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the TNF-gamma genes. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

[0131] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0132] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0133] The DNA sequence in the expression vector is operably associatedwith an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli lac or trp, the phagelambda P promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0134] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase, glutaminesynthase, or neomycin resistance for eukaryotic cell culture, or such astetracycline or ampicillin resistance in E. coli.

[0135] Vectors which use glutamine synthase (GS) or DHFR as theselectable markers can be amplified in the presence of the drugsmethionine sulphoximine or methotrexate, respectively. The availabilityof drugs which inhibit the function of the enzymes encoded by theseselectable markers allows for selection of cell lines in which thevector sequences have been amplified after integration into the hostcell's DNA. An advantage of glutamine synthase based vectors are theavailability of cell lines (e.g., the murine myeloma cell line, NSO)which are glutamine synthase negative. Glutamine synthase expressionsystems can also function in glutamine synthase expressing cells (e.g.Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor toprevent the functioning of the endogenous gene. A glutamine synthaseexpression system and components thereof are detailed in PCTpublications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are hereby incorporated in their entireties byreference herein. Additionally, glutamine synthase expression vectorsthat may be used according to the present invention are commerciallyavailable from suppliers including, for example, Lonza Biologics, Inc.(Portsmouth, N.H.). Expression and production of monoclonal antibodiesusing a GS expression system in murine myeloma cells is described inBebbington et al., Bio/technology 10:169(1992) and in Biblia andRobinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated byreference.

[0136] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0137] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Sf9; animal cells such as CHO, NSO, COS or Bowesmelanoma, adenoviruses, plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0138] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably associated with thesequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, and are commercially available. The followingvectors are provided by way of example. Bacterial: pHE4-5 (ATCCAccession No. 209311; and variations thereof), pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any otherplasmid or vector may be used as long as they are replicable and viablein the host.

[0139] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacl, lacZ, T3, T7, gpt, lambda P, P, andtrp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retroviruses, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0140] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation. (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0141] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., TNF-gamma coding sequence),and/or to include genetic material (e.g., heterologous polynucleotidesequences) that is operably associated with TNF-gamma polynucleotides ofthe invention, and which activates, alters, and/or amplifies endogenousTNF-gamma polynucleotides. For example, techniques known in the art maybe used to operably associate heterologous control regions (e.g.,promoter and/or enhancer) and endogenous TNF-gamma polynucleotidesequences via homologous recombination (see, e.g., U.S. Pat. No.5,641,670, issued Jun. 24, 1997; International Publication No. WO96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989),the disclosures of each of which are incorporated by reference in theirentireties).

[0142] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0143] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0144] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0145] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, for example, stabilization or simplifiedpurification of expressed recombinant product.

[0146] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0147] As a representative, but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0148] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. Cells aretypically harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification.

[0149] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0150] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman (Cell 23:175 (1981)), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0151] The TNF-gamma polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0152] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0153] The invention encompasses TNF-gamma-alpha and TNF-gamma-betapolypeptides which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited, to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

[0154] The present invention further encompasses encompassesTNF-gamma-alpha and/or TNF-gamma-beta polypeptides or fragments thereofconjugated to a diagnostic agent (e.g. a detectable agent) and/ortherapeutic agent. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance may becoupled or conjugated either directly to the polypeptide (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. See, forexample, U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto polypeptides for use as diagnostics and/or therapeutics according tothe present invention. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium(⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorline (¹⁸F), ¹⁵³Sm,¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re,¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru. A preferred radioisotope label is ¹¹¹I. Anotherpreferred radioactive label is ⁹⁰Y. Another preferred radioactive labelis ¹³¹I.

[0155] Further, a TNF-gamma-alpha and/or TNF-gamma-beta polypeptide orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters such as, for example,²¹³Bi or other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I,⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, ⁹⁰Y, ¹¹⁷Tin, ¹⁸⁶Re, ¹⁸⁸Re and ¹⁶⁶Ho. In specificembodiments, an antibody or fragment thereof is attached to macrocyclicchelators useful for conjugating radiometal ions, including but notlimited to, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In specificembodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the polypeptide ofthe invention or fragment thereof via a linker molecule. Examples oflinker molecules useful for conjugating DOTA to a polypeptide arecommonly known in the art—see, for example, DeNardo et al., Clin CancerRes. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7,1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which arehereby incorporated by reference in their entirety.

[0156] A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).^(The conjugates of the invention may also be used to target and destroy specific cell types, such as T cells, particularly cancerous T cells. Thus, the present invention further encompasses methods and compositions for killing cells of hematopoietic origin, comprising, or alternatively consisting of, contacting TNF-gamma alpha and/or TNF-gamma-beta polypeptide or fragment or variant thereof (e.g., TNF-gamma alpha and/or TNF-gamma-beta polypeptide or fragment or variant thereof conjugated to a radioisotope, cytotoxin or cytotoxic pro-drug) with cells of hematopoietic origin. In preferred embodiments, the cells of hematopoietic origin are T cells. In other non-exclusive preferred embodiments, the cells of hematopoietic origin are cancerous T cells.)

[0157] The present invention further encompasses methods andcompositions for killing cells of hematopoietic origin, comprising, oralternatively consisting of, administering to an animal, preferably ahuman, in which such killing of hematopoietic cells is desired, aTNF-gamma alpha and/or TNF-gamma-beta polypeptide or fragment or variantthereof (e.g., TNF-gamma alpha and/or TNF-gamma-beta polypeptide orfragment or variant thereof conjugated to a radioisotope, cytotoxin orcytotoxic pro-drug) in an amount effective to kill cells ofhematopoietic origin. In preferred embodiments, the cells ofhematopoietic origin are T cells. In preferred embodiments, the cells ofhematopoietic origin are cancerous T cells.

[0158] Techniques known in the art may be applied to label polypeptidesand antibodies (as well as fragments and variants of polypeptides andantibodies) of the invention. Such techniques include, but are notlimited to, the use of bifunctional conjugating agents (see e.g., U.S.Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931;5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and5,808,003; the contents of each of which are hereby incorporated byreference in its entirety) and direct coupling reactions (e.g.,Bolton-Hunter and Chloramine-T reaction).

[0159] In addition, polypeptides of the invention can be chemicallysynthesized using techniques known in the art. For example, a peptidecorresponding to a fragment of the TNF-gamma-alpha or TNF-gamma-betapolypeptides of the invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the sequence. Non-classical amino acids include but arenot limited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

[0160] At least fifteen TNF-gamma-alpha expression constructs have beengenerated by the inventors herein to facilitate the production ofTNF-gamma polypeptides of several sizes and in several systems. Ofthese, four have been constructed which encode a full-length TNF-gammapolypeptide. The full-length constructs are: (i) pQE9TNFg-27/147, (ii)pQE70TNFg, (iii) pC1TNFg, and pcDNA3TNFg. In the case of the firstexpression construct listed (pQE9TNFg-27/147), the construct was used toproduce a full-length TNF-gamma-alpha polypeptide with an N-terminal sixhistidine amino acid tag according to the method of Example 1. Afull-length TNF-gamma-alpha polypeptide lacking the histidine tag wasproduced in bacteria by using the pQE70TNFg construct essentially as wasdone in Example 1. In addition, a full-length TNF-gamma-alphapolypeptide lacking a histidine tag was produced in mammalian cells byusing either the pC1TNFg or pcDNA3TNFg constructs according to themethod of Example 3. Further, the mature TNF-gamma-alpha polypeptide wasproduced and secreted from mammalian cells under direction of theinterleukin (IL)-6 signal peptide from a construct designatedpcDNA3/IL6TNFg-1/149 (see Example 11).

[0161] The remaining TNF-gamma-alpha expression constructs were used toexpress various TNF-gamma muteins from bacterial, baculoviral, andmammalian systems. Four N-terminal deletion mutations have beengenerated using the pQE60 bacterial expression vector. These N-terminaldeletion mutation constructs are: (i) pQE60TNFg-3/147 (representing apossible mature TNF-gamma polypeptide; the polypeptide expressed by thisconstruct is identical to amino acid residues 107-251 of theTNF-gamma-beta of SEQ ID NO:20), (ii) pQE60TNFg12/147 (representingamino acid residues 12-147 of SEQ ID NO:2 and residues 116-251 of SEQ IDNO:20), (iii) pQE60TNFg22/147 (representing amino acid residues 22-147of SEQ ID NO:2 and residues 126-251 of SEQ ID NO:20), and (iv)pQE60TNFg28/147 (representing amino acid residues 28-147 of SEQ ID NO:2and residues 132-251 of SEQ ID NO:20). Each of these expressionconstructs can be used to produce a TNF-gamma polypeptide in a bacterialsystem which exhibits an N-terminal deletion of 24, 38, 48, and 54 aminoacids, respectively, with regard to the full-length TNF-gamma-alphapolypeptide or an N-terminal deletion of 106, 115, 125, and 131 aminoacids, respectively, with regard to the full-length TNF-gamma-betapolypeptide.

[0162] Further N-terminal deletion mutation bacterial expressionconstructs have been generated. A construct designated pHE4 VEGIT30-L174 has been generated using the bacterial expression vector pHE4to express amino acids threonine-30 to leucine-174 of theTNF-gamma-alpha sequence shown in FIGS. 1A and 1B (residues threonine-3to leucine-147 of SEQ ID NO:2) which correspond exactly to amino acidresidues threonine-107 to leucine-251 of the TNF-gamma-beta sequenceshown in FIGS. 20A and 20B (residues threonine-107 to leucine-251 of SEQID NO:20). Additional bacterial expression constructs generated includepQE9.VEGI.his.T28-L174, pHE4.VEGI.T28-L174, pHE4.VEGI.T51-L174, andpHE4.VEGI.T58-L174. These constructs are based on either the pQE9 orpHE4 bacterial expression vectors. The construct designations indicatethe expression vector, the gene name, and the amino acid residuesexpressed by the construct (e.g. pQE9.VEGI.T28-L174 indicates that thepQE9 bacterial expression vector is used to express amino acidsthreonine (T)-28 through leucine (L)-174 of the TNF-gamma-alphapolypeptide (VEGI is a laboratory designation for TNF-gamma-alpha)).

[0163] A TNF-gamma expression construct has been generated which can beused to produce a secreted mature TNF-gamma polypeptide from a mammaliansystem. The construct has been designated pC1/IL6TNFg-3/147. It encodesthe signal peptide from the human IL-6 gene fused to the matureTNF-gamma sequence. A similar construct has been generated whichcontains the CK-beta8 signal peptide (amino acids -21 to -1 of theCK-beta8 sequence disclosed in published PCT application PCT/US95/09058;filed Jun. 23, 1995) fused to the amino terminus of amino acids 12-149of TNF-gamma-alpha (SEQ ID NO:2; that is, amino acids 116-251 ofTNF-gamma-beta (SEQ ID NO:20)) in the context of the pC4 mammalianexpression vector. This construct has been designatedpC4/CK-beta8TNFg12/147. A variant of this construct has been generatedwhich can be used to express amino acids 12-147 of TNF-gamma fused tothe human IgG Fc region at the TNF-gamma carboxy terminus. This fusionprotein is also secreted under the direction of the CK-beta8 signalpeptide and has been designated pC4/CK-beta8TNFg12/147/Fc. The sequenceof the human Fc portion of the fusion molecule is shown in SEQ ID NO:18.Other sequences could be used which are known to those of skill in theart.

[0164] Amino acids -3 to 147 of TNF-gamma-alpha (SEQ ID NO:2; whichcorrespond to amino acid residues 102 to 251 of TNF-gamma-beta (SEQ IDNO:20)) can be expressed and secreted from a baculovirus system by usinga construct designated pA2GPTNFg-3/147. This expression constructencodes the mature TNF-gamma coding sequence fused at its amino terminusto the baculoviral GP signal peptide.

[0165] Two retroviral TNF-gamma expression constructs have also beengenerated. The first of these has been designated pG1SamEN/TNFg-3/149.This expression construct can be used to produce full-length TNF-gammaprotein from a mammalian system. A related construct,pG1SamEN/CK-beta8TNFg12/149, has been generated which can be used toproduce and secrete mature TNF-gamma protein from a mammalian systemunder the direction of the CK-beta8 signal peptide.

[0166] Further polypeptides of the present invention includepolypeptides which have at least 80%, 85% or 90% similarity, morepreferably at least 92%, 94% or 95% similarity, and still morepreferably at least 96%, 97%, 98% or 99% similarity to those describedabove. The polypeptides of the invention also comprise those which areat least 80% or 85% identical, more preferably at least 90%, 92%, 94% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

[0167] In preferred embodiments, polynucleotides of the inventioninclude nucleotides 1-543 (or 4-543 if the vector supplies anamino-terminal ATG) of SEQ ID NO:25 inserted in-frame into any of theexpression constructs described herein (such, for example, pHE4, pHE4b,pHE4-5, pA2, pA2GP, pC4, pC4/CK-beta8, pG1SamEN, pG1SamEN/CK-beta8,etc.). Polypeptides encoded by these polynucleotides are alsoencompassed by the invention. The present invention is also directed tonucleic acid molecules comprising, or alternatively, consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%,98% or 99% identical to the polynucleotide sequences encoding theTNF-gamma-beta polypeptides described above, and the polypeptidesencoded thereby. The present invention also encompasses the abovepolynucleotide sequences fused to a heterologous polynucleotide sequence(such as, for example, a polynucleotide sequence encoding the Fc regionof a human immunoglobulin or FLAG (see, for example, Example 16)), andthe polypeptides encoded thereby.

[0168] In additional preferred embodiments, polynucleotides of theinvention include nucleotides 214-753 of SEQ ID NO:20 inserted in-frameinto any of the expression constructs described herein (such, forexample, pHE4, pHE4b, pHE4-5, pA2, pA2GP, pC4, pC4/CK-beta8, pG1SamEN,pG1SamEN/CK-beta8, etc.). In these embodiments, the vector or theTNF-gamma-beta polynucleotide of the invention may contribute anamino-terminal ATG codon. Polypeptides encoded by these polynucleotidesare also encompassed by the invention. The present invention is alsodirected to nucleic acid molecules comprising, or alternatively,consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequencesencoding the TNF-gamma-beta polypeptides described above, and thepolypeptides encoded thereby. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence (such as, for example, a polynucleotide sequence encoding theFc region of a human immunoglobulin or FLAG (see, for example, Example16)), and the polypeptides encoded thereby.

Polypeptides and Fragments

[0169] The present invention further relates to an isolatedTNF-gamma-alpha polypeptide which has the deduced amino acid sequence ofFIGS. 1A and 1B (SEQ ID NO:2) or which has the amino acid sequenceencoded by the deposited cDNA HUVEO91, as well as fragments, analogs andderivatives of such polypeptide.

[0170] The present invention also relates to a TNF-gamma-betapolypeptide which has the deduced amino acid sequence of FIGS. 20A and20B (SEQ ID NO:20) or which has the amino acid sequence encoded by thedeposited cDNA HEMCZ56, as well as fragments, analogs and derivatives ofsuch polypeptide.

[0171] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified toa point within the range of near complete (e.g., >90% pure) to complete(e.g., >99% pure) homogeneity. The term “isolated” means that thematerial is removed from its original environment (e.g., the naturalenvironment if it is naturally occurring). For example, anaturally-occurring polynucleotide or polypeptide present in a livinganimal is not isolated, but the same polynucleotide or polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. Also intended as an “isolated polypeptide” arepolypeptides that have been purified partially or substantially from arecombinant host cell. For example, a recombinantly produced version ofa TNF-gamma polypeptide can be substantially purified by the one-stepmethod described by Smith and Johnson (Gene 67:31-40 (1988)). Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.Isolated polypeptides and polynucleotides according to the presentinvention also include such molecules produced naturally orsynthetically. Polypeptides and polynucleotides of the invention alsocan be purified from natural or recombinant sources using anti-TNF-gammaantibodies of the invention in methods which are well known in the artof protein purification.

[0172] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptides of FIGS. 1A and 1B or FIGS. 20A and 20B, and thosepolypeptides encoded by the deposited cDNAs, means a polypeptide whichretains a TNF-gamma functional activity, i.e., displays one or morefunctional activities associated with a full-length and/or matureTNF-gamma polypeptide disclosed in FIGS. 1A and B (SEQ ID NO:2), FIGS.20A and B (SEQ ID NO:20), disclosed elsewhere herein, and/or encoded byone or both of the deposited clones (HUVEO91 and HEMCZ56). As oneexample, such fragments, derivatives, or analogs, which have the desiredimmunogenicity or antigenicity can be used, for example, inimmunoassays, for immunization, for inhibition of TNF-gamma activity,etc. Thus, a specific embodiment of the invention relates to a TNF-gammafragment that can be bound by an antibody that specifically binds theTNF-gamma polypeptide sequence disclosed in FIGS. 1A and B (SEQ IDNO:2), FIGS. 20 A and B (SEQ ID NO:20) ), and/or which is encoded by oneor both of the deposited clones (HUVEO91 and HEMCZ56).

[0173] As another example, TNF-gamma fragments, derivatives or analogswhich have TNF-gamma biological activity (e.g., a mature TNF-gamma-alphapolypeptide or the extracellular domain of a TNF-gamma-beta polypeptide)are provided. TNF-gamma fragments, derivatives, and analogs that retain,or alternatively lack a desired TNF-gamma property of interest (e.g.,inhibition of cell proliferation, tumor inhibition, inhibition ofangiogenesis, anti-arthritis by the inhibition of angiogenesis and/orendothelial cell proliferation associated with invading pannus in boneand cartilage, an inducer of NF-kappaB and c-Jun kinase (JNK), aninducer of cell adhesion, and as an inducer apoptosis (See Examples,particularly Examples 12-15)) can be used as inducers or inhibitors,respectively, of such properties and its physiological correlates.

[0174] The polypeptides of the invention may exist as a membrane boundreceptor having a transmembrane region and an intra- and extracellularregion or they may exist in soluble form wherein the transmembranedomain is lacking. One example of such a form of TNF-gamma is theTNF-gamma-beta polypeptide sequence shown in FIGS. 20A and B (SEQ IDNO:20) which contains a transmembrane, intracellular and extracellulardomain, as described herein.

[0175] It will be recognized in the art that some amino acid sequencesof the TNF-gamma polypeptide can be varied without significant effect ofthe structure or function of the protein. If such differences insequence are contemplated, it should be remembered that there will becritical areas on the protein which determine activity. Thus, theinvention further includes variations of the TNF-gamma polypeptide whichshow substantial TNF-gamma polypeptide activity or which include regionsof TNF-gamma protein such as the polypeptide fragments disclosed herein.Such variants include deletions, insertions, inversions, repeats, andtype substitutions selected according to general rules known in the artso as have little effect on activity. For example, guidance concerninghow to make phenotypically silent amino acid substitutions is providedwherein the authors indicate that there are two main approaches forstudying the tolerance of an amino acid sequence to change (Bowie etal., Science 247:1306-1310 (1990)). The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described by Bowie and coworkers (supra) and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

[0176] Thus, the polypeptide of SEQ ID NO:2, or of SEQ ID NO:20, or thefragment, derivative or analog of the polypeptide of SEQ ID NO:2, or ofSEQ ID NO:20, or the polypeptides encoded by the deposited cDNAs, may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, or (ii) one in whichone or more of the amino acid residues includes a substituent group, or(iii) one in which the mature form of the TNF-gamma polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the above form of thepolypeptide, such as an IgG Fc peptide, human serum albumin or afragment or variant thereof (see, e.g., U.S. Pat. No. 5,876,969, issuedMar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issuedJun. 16, 1998, herein incorporated by reference in their entirety)), aleader or secretory sequence, or a sequence which is employed forpurification of the above form of the polypeptide or a proproteinsequence. Such fragments, derivatives and analogs are deemed to bewithin the scope of those skilled in the art from the teachings herein.

[0177] Thus, the TNF-gamma of the present invention may include one ormore amino acid substitutions, deletions or additions, either fromnatural mutations or human manipulation. As indicated, changes arepreferably of a minor nature, such as conservative amino acidsubstitutions that do not significantly affect the folding or activityof the protein (see Table 1). TABLE 1 Conservative Amino AcidSubstitutions. Aromatic Phenylalanine Tryptophan Tyrosine HydrophobicLeucine Isoleucine Valine Polar Glutamine Asparagine Basic ArginineLysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine SerineThreonine Methionine Glycine

[0178] Embodiments of the invention are directed to polypeptides whichcomprise the amino acid sequence of a TNF-gamma polypeptide describedherein, but having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, even more preferably, not more than 40conservative amino acid substitutions, still more preferably, not morethan 30 conservative amino acid substitutions, and still even morepreferably, not more than 20 conservative amino acid substitutions, whencompared with the TNF-gamma polynucleotide sequence described herein. Ofcourse, in order of ever-increasing preference, it is highly preferablefor a peptide or polypeptide to have an amino acid sequence whichcomprises the amino acid sequence of a TNF-gamma polypeptide, whichcontains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

[0179] In further specific embodiments, the number of substitutions,additions or deletions in the amino acid sequence of FIGS. 1A and B (SEQID NO:2), FIGS. 20A and B (SEQ ID NO:20), a polypeptide sequence encodedby the deposited clones, and/or any of the polypeptide fragmentsdescribed herein (e.g., the extracellular domain or intracellulardomain) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4,3, 2, 1 or 150-50, 100-50, 50-20, 30-20, 20-15, 20-10, 15-10, 10-1,5-10, 1-5, 1-3 or 1-2.

[0180] To improve or alter the characteristics of TNF-gammapolypeptides, protein engineering may be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelmutant proteins or muteins including single or multiple amino acidsubstitutions, deletions, additions or fusion proteins. Such modifiedpolypeptides can show, e.g., enhanced activity or increased stability.In addition, they may be purified in higher yields and show bettersolubility than the corresponding natural polypeptide, at least undercertain purification and storage conditions.

[0181] Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

[0182] Thus, the invention also encompasses TNF-gamma derivatives andanalogs that have one or more amino acid residues deleted, added, orsubstituted to generate TNF-gamma polypeptides that are better suitedfor expression, scale up, etc., in the host cells chosen. For example,cysteine residues can be deleted or substituted with another amino acidresidue in order to eliminate disulfide bridges; N-linked glycosylationsites can be altered or eliminated to achieve, for example, expressionof a homogeneous product that is more easily recovered and purified fromyeast hosts which are known to hyperglycosylate N-linked sites. To thisend, a variety of amino acid substitutions at one or both of the firstor third amino acid positions on any one or more of the glycosylationrecognitions sequences in the TNF-gamma polypeptides of the invention,and/or an amino acid deletion at the second position of any one or moresuch recognition sequences will prevent glycosylation of the TNF-gammapolypeptide at the modified tripeptide sequence (see, e.g., Miyajimo etal., EMBO J 5(6):1193-1197).

[0183] Additionally, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities ofTNF-gamma-alpha and/or TNF-gamma-beta thereby effectively generatingagonists and antagonists of TNF-gamma-alpha and/or TNF-gamma-beta. Seegenerally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol.8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998);Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M.M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference). In oneembodiment, alteration of TNF-gamma-alpha and/or TNF-gamma-betapolynucleotides and corresponding polypeptides may be achieved by DNAshuffling. DNA shuffling involves the assembly of two or more DNAsegments into a desired TNF-gamma-alpha and/or TNF-gamma-beta moleculeby homologous, or site-specific, recombination. In another embodiment,TNF-gamma-alpha and/or TNF-gamma-beta polynucleotides and correspondingpolypeptides may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of TNF-gamma-alpha and/orTNF-gamma-beta may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules are, forexample, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, AIM-I (InternationalPublication No. WO 97/33899), AIM-II (International Publication No. WO97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha(International Publication No. WO 98/07880), Neutrokine-alpha(International Publication No. WO 98/18921), OPG, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694),TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892, TRIO (International Publication No. WO98/54202),312C2 (International Publication No. WO 98/06842), TR11,TR11SV1, TR11SV2, TR12, and TNF-R1, TRAMP/DR3/APO-3/WSL/LARD, TRAIL-R1/DR4/APO-2, TRAIL-R2/DR5, DcR 1/TRAIL-R3/TRID/LIT, DcR2/TRAIL-R4, CAD,TRAIL, TRAMP, v-FLIP.

[0184] In further preferred embodiments, the heterologous molecules areany member of the TNF family.

[0185] In a preferred embodiment, the compositions of the invention areadministered in combination with CD40 ligand (CD40L), a soluble form ofCD40L (e.g., AVREND™), biologically active fragments, variants, orderivatives of CD40L, anti-CD40L antibodies (e.g.,. agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonisticor antagonistic antibodies).

[0186] Amino acids in the TNF-gamma protein of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as receptor binding or in vitro proliferativeactivity.

[0187] Of special interest are substitutions of charged amino acids withother charged or neutral amino acids which may produce proteins withhighly desirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins, et al., Diabetes 36:838-845 (1987); Cleland, et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

[0188] Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

[0189] Since TNF-gamma is a member of the TNF-related protein family, tomodulate rather than completely eliminate biological activities ofTNF-gamma preferably additions, substitutions, or deletions are made insequences encoding amino acids in the conserved TNF-like domain, i.e.,in positions 17-147 of SEQ ID NO:2 or positions 121-251 of SEQ ID NO:20,more preferably in residues within this region which are not conservedin all members of the TNF-related protein family (see FIGS. 2A-2C). Alsoforming part of the present invention are isolated polynucleotidescomprising nucleic acid sequences which encode the above TNF-gammavariants.

[0190] Several amino acids of the TNF-gamma polypeptide are highlyconserved across the known members of the TNF-related protein family. Bymaking a specific mutation in TNF-gamma in such residues astryptophan-15 (as numbered in SEQ ID NO:2), leucine-35, glycine-41,tyrosine-43, tyrosine-46, glutamine-48, leucine-90, leucine-116,glycine-119, aspartic acid-120, phenylalanine-141, phenylalanine-142,and leucine-147, it is likely that a noticeable effect on biologicalactivity will be observed. These identical amino acid residues are, ofcourse, present in the corresponding positions of TNF-gamma-beta shownin SEQ ID NO:20.

[0191] The present invention also encompasses fragments of theabove-described TNF-gamma polypeptides. Polypeptide fragments of thepresent invention include polypeptides comprising an amino acid sequencecontained in SEQ ID NO:2, encoded by the cDNA contained in the depositedclone (HUVEO91), or encoded by nucleic acids which hybridize (e.g. understringent hybridization conditions) to the nucleotide sequence containedin the deposited clones, that shown in FIGS. 1A and 1B (SEQ ID NO:1)and/or FIGS. 20A and 20B (SEQ ID NO:19), or the complementary strandthereto.

[0192] Polypeptide fragments may be “free-standing” or comprised withina larger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, included, for example, fragmentsthat comprise or alternatively, consist of, from about amino acidresidues, 1 to 20, 21 to 40, 41 to 60, 61 to 83, 84 to 100, 101 to 120,121 to 140, 141 to 160, 160 to 167, 161 to 174, 161 to 180, 181 to 200,201 to 220, 221 to 240, 241 to 251 of SEQ ID NO:2 and/or SEQ ID NO:20.Moreover, polypeptide fragments can be at least about 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids in length. Inthis context “about” includes the particularly recited ranges, larger orsmaller by several (i.e. 5, 4, 3, 2 or 1) amino acids, at either extremeor at both extremes.

[0193] In other embodiments, the fragments or polypeptides of theinvention (i.e., those described herein) are not larger than 250, 225,200, 185, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120,115, 110, 105, 100, 90, 80, 75, 60, 50, 40, 30 or 25 amino acidsresidues in length.

[0194] Further preferred embodiments encompass polypeptide fragmentscomprising, or alternatively consisting of, the mature domain ofTNF-gamma-alpha (amino acid residues 1-147 of SEQ ID NO:2), theintracellular domain of TNF-gamma-beta (amino acid residues 1-35 of SEQID NO:20), the transmembrane domain of TNF-gamma-beta (amino acidresidues 36-61 of SEQ ID NO:20), the transmembrane domain ofTNF-gamma-beta (amino acid residues 36-59 of SEQ ID NO:20), theextracellular domain of TNF-gamma-beta (amino acid residues 62-251 ofSEQ ID NO:20), and/or the extracellular domain of TNF-gamma-beta (aminoacid residues 60-251 of SEQ ID NO:20).

[0195] In specific embodiments, polypeptide fragments of the inventioncomprise, or alternatively, consist of, amino acid residues leucine-35to valine-49, tryptophan-104 to leucine-116, glycine-119 to serine-127,lysine-139 to leucine-147 of SEQ ID NO:2). These domains are regions ofhigh identity identified by comparison of the TNF family memberpolypeptides shown in FIGS. 2A, 2B, and 2C.

[0196] Among the especially preferred fragments of the invention arefragments characterized by structural or functional attributes ofTNF-gamma. Such fragments include amino acid residues that comprisealpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet-forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., regions of polypeptidesconsisting of amino acid residues having an antigenic index of or equalto greater than 1.5, as identified using the default parameters of theJameson-Wolf program) of TNF-gamma. Certain preferred regions are thosedisclosed in FIG. 17 and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequencedepicted in FIGS. 1A and B, such preferred regions include;Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Chou-Fasman predicted alpha-regions, beta-regions,turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic andhydrophobic regions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0197] Additionally, analogs of the invention include a proprotein whichcan be activated by cleavage of the proprotein portion to produce anactive mature polypeptide.

[0198] In another embodiment, the invention provides a TNF-gammapolypeptide (e.g., fragment) comprising, or alternatively, consistingof, an epitope-bearing portion of a polypeptide of the invention. Theepitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope”. The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes (see, forinstance, Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983)).

[0199] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein (see, for instance, Sutcliffe, J.G., et al., Science 219:660-666 (1983)). Peptides capable of elicitingprotein-reactive sera are frequently represented in the primary sequenceof a protein, can be characterized by a set of simple chemical rules,and are confined neither to immunodominant regions of intact proteins(i.e., immunogenic epitopes) nor to the amino or carboxyl termini.Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention (see, forinstance, Wilson, et al., Cell 37:767-778 (1984)).

[0200] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between about 15 to about30 amino acids contained within the amino acid sequence of a polypeptideof the invention. Non-limiting examples of antigenic polypeptides orpeptides that can be used to generate TNF-gamma-specific antibodiesinclude: a polypeptide comprising amino acid residues from about Thr-24to about Asn-32 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about Ile-37 to about Ile-45 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about Met-54 to about Arg-62 in SEQID NO:2; a polypeptide comprising amino acid residues from about Gln-63to about Asp-71 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about Glu-57 to about Gly-65 in SEQ ID NO:2; a polypeptidecomprising amino acid residues from about Val-80 to about Thr-88 in SEQID NO:2; a polypeptide comprising amino acid residues from about Leu-116 to about Val-124 in SEQ ID NO:2; and a polypeptide comprising aminoacid residues from about Asp-133 to about Phe-141 in SEQ ID NO:2. Thesepolypeptide fragments have been determined to bear antigenic epitopes ofthe TNF-gamma protein by the analysis of the Jameson-Wolf antigenicindex, as shown in FIG. 17, above.

[0201] One of ordinary skill in the art may easily determine antigenicregions for TNF-gamma-beta by using data prepared through a DNA*STARanalysis of the TNF-gamma-beta polypeptide sequence (SEQ ID NO:20) usingthe default parameters and selecting regions with a high antigenic indexas described above.

[0202] In another aspect, the invention provides peptides andpolypeptides comprising epitope-bearing portions of the polypeptides ofthe present invention. These epitopes are immunogenic or antigenicepitopes of the polypeptides of the present invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse in vivo when the whole polypeptide of the present invention, orfragment thereof, is the immunogen. On the other hand, a region of apolypeptide to which an antibody can bind is defined as an “antigenicdeterminant” or “antigenic epitope.” The number of in vivo immunogenicepitopes of a protein generally is less than the number of antigenicepitopes. See, e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA81:3998-4002. However, antibodies can be made to any antigenic epitope,regardless of whether it is an immunogenic epitope, by using methodssuch as phage display. See e.g., Petersen G. et al. (1995) Mol. Gen.Genet. 249:425-431. Therefore, included in the present invention areboth immunogenic epitopes and antigenic epitopes.

[0203] A list of exemplified amino acid sequences comprising immunogenicepitopes is described above. It is pointed out that the list ofimmunogenic epitopes only lists amino acid residues comprising epitopespredicted to have the highest degree of antigenicity using the algorithmof Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186 (saidreferences incorporated by reference in their entireties). TheJameson-Wolf antigenic analysis was performed using the computer programPROTEAN, using default parameters (Version 3.11 for the Power MacIntosh,DNASTAR, Inc., 1228 South Park Street Madison, Wis.). Portions ofpolypeptides not listed in the above list of immunogenic epitopes arenot considered non-immunogenic. The immunogenic epitopes listed above isan exemplified list, not an exhaustive list, because other immunogenicepitopes are merely not recognized as such by the particular algorithmused. Amino acid residues comprising other immunogenic epitopes may beroutinely determined using algorithms similar to the Jameson-Wolfanalysis or by in vivo testing for an antigenic response using methodsknown in the art. See, e.g., Geysen et al., supra; U.S. Pat. Nos.4,708,781; 5,194,392; 4,433,092; and 5,480,971 (said referencesincorporated by reference in their entireties).

[0204] It is particularly pointed out that the amino acid sequenceslisted above comprise immunogenic epitopes. The list of immunogenicepitopes lists only the critical residues of immunogenic epitopesdetermined by the Jameson-Wolf analysis. Thus, additional flankingresidues on either the N-terminal, C-terminal, or both N- and C-terminalends may be added to the sequences listed above to generate anepitope-bearing polypeptide of the present invention. Therefore, theimmunogenic epitopes listed above may include additional N-terminal orC-terminal amino acid residues. The additional flanking amino acidresidues may be contiguous flanking N-terminal and/or C-terminalsequences from the polypeptides of the present invention, heterologouspolypeptide sequences, or may include both contiguous flanking sequencesfrom the polypeptides of the present invention and heterologouspolypeptide sequences.

[0205] Polypeptides of the present invention comprising immunogenic orantigenic epitopes are at least 7 amino acids residues in length. “Atleast” means that a polypeptide of the present invention comprising animmunogenic or antigenic epitope may be 7 amino acid residues in lengthor any integer between 7 amino acids and the number of amino acidresidues of the full length polypeptides of the invention. Preferredpolypeptides comprising immunogenic or antigenic epitopes are at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 amino acid residues in length. However, it is pointed out thateach and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.

[0206] The immuno and antigenic epitope-bearing fragments may bespecified by either the number of contiguous amino acid residues, asdescribed above, or further specified by N-terminal and C-terminalpositions of these fragments on the amino acid sequence of SEQ ID NO:2.Every combination of a N-terminal and C-terminal position that afragment of, for example, at least 7 or at least 15 contiguous aminoacid residues in length could occupy on the amino acid sequence of SEQID NO:2 is included in the invention. Again, “at least 7 contiguousamino acid residues in length” means 7 amino acid residues in length orany integer between 7 amino acids and the number of amino acid residuesof the full length polypeptide of the present invention. Specifically,each and every integer between 7 and the number of amino acid residuesof the full length polypeptide are included in the present invention.Further, immuno- and antigenic epitope-bearing fragments may bespecified in the same way for TNF-gamma-beta by using the techniquesdescribed herein.

[0207] Immunogenic and antigenic epitope-bearing polypeptides of theinvention are useful, for example, to make antibodies which specificallybind the polypeptides of the invention, and in immunoassays to detectthe polypeptides of the present invention. The antibodies are useful,for example, in affinity purification of the polypeptides of the presentinvention. The antibodies may also routinely be used in a variety ofqualitative or quantitative immunoassays, specifically for thepolypeptides of the present invention using methods known in the art.See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press; 2nd Ed. 1988).

[0208] The epitope-bearing polypeptides of the present invention may beproduced by any conventional means for making polypeptides includingsynthetic and recombinant methods known in the art. For instance,epitope-bearing peptides may be synthesized using known methods ofchemical synthesis. For instance, Houghten has described a simple methodfor the synthesis of large numbers of peptides, such as 10-20 mgs of 248individual and distinct 13 residue peptides representing single aminoacid variants of a segment of the HA1 polypeptide, all of which wereprepared and characterized (by ELISA-type binding studies) in less thanfour weeks (Houghten, R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135(1985)). This “Simultaneous Multiple Peptide Synthesis (SMPS)” processis further described in U.S. Pat. No. 4,631,211 to Houghten andcoworkers (1986). In this procedure the individual resins for thesolid-phase synthesis of various peptides are contained in separatesolvent-permeable packets, enabling the optimal use of the manyidentical repetitive steps involved in solid-phase methods. A completelymanual procedure allows 500-1000 or more syntheses to be conductedsimultaneously (Houghten et al. (1985) Proc. Natl. Acad. Sci.82:5131-5135 at 5134.

[0209] Epitope-bearing polypeptides of the present invention are used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe, et al., supra; Wilson, etal., supra, and Bittle, et al. (1985) J. Gen. Virol. 66:2347-2354. If invivo immunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling of thepeptide to a macromolecular carrier, such as keyhole limpet hemocyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such as-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carriers using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μgs of peptide or carrier protein and Freund's adjuvant.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0210] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention (e.g., those comprisingan immunogenic or antigenic epitope) can be fused to heterologouspolypeptide sequences. For example, polypeptides of the presentinvention (including fragments or variants thereof), may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof,resulting in chimeric polypeptides. By way of another non-limitingexample, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused with albumin(including but not limited to recombinant human serum albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). In a preferred embodiment, polypeptides and/or antibodies ofthe present invention (including fragments or variants thereof) arefused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322094) which is herein incorporated by reference in its entirety. Inanother preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-z of human serum albumin, where z is an integerfrom 369 to 419, as described in U.S. Pat. No. 5,766,883 hereinincorporated by reference in its entirety. Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N- or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

[0211] Such fusion proteins as those described above may facilitatepurification, and show an increased half-life in vivo. This has beenshown, e.g., for chimeric proteins consisting of the first two domainsof the human CD4-polypeptide and various domains of the constant regionsof the heavy or light chains of mammalian immunoglobulins. See, e.g.,EPA 0,394,827; Traunecker et al. (1988) Nature 331:84-86. Fusionproteins that have a disulfide-linked dimeric structure due to the IgGportion can also be more efficient in binding and neutralizing othermolecules than monomeric polypeptides or fragments thereof alone. See,e.g., Fountoulakis et al. (1995) J. Biochem. 270:3958-3964. Nucleicacids encoding the above epitopes can also be recombined with a gene ofinterest as an epitope tag to aid in detection and purification of theexpressed polypeptide.

[0212] The epitope-bearing peptides and polypeptides of the produced byany conventional means (see, for example, Houghten, R. A., et al., Proc.Natl. Acad. Sci. USA 82:5131-5135 (1985); and U.S. Pat. No. 4,631,211 toHoughten, et al. (1986)).

[0213] Epitope-bearing peptides and polypeptides of the inventioninvention have uses which include, but are not limited to, inducingantibodies according to methods well known in the art (see, forinstance, Sutcliffe, et aL, supra; Wilson, et aL, supra; Chow, M., etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J., et al, JGen. Virol. 66:2347-2354 (1985)). Immunogenic epitope-bearing peptidesof the invention, i.e., those parts of a protein that elicit an antibodyresponse when the whole protein is the immunogen, are identifiedaccording to methods known in the art (see, for instance, Geysen, et al,supra). Further still, U.S. Pat. No. 5,194,392, issued to Geysen,describes a general method of detecting or determining the sequence ofmonomers (amino acids or other compounds) which is a topologicalequivalent of the epitope (i.e., a “mimotope”) which is complementary toa particular paratope (antigen binding site) of an antibody of interest.More generally, U.S. Pat. No. 4,433,092, issued to Geysen, describes amethod of detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest (e.g. DR3).Similarly, U.S. Pat. No. 5,480,971, issued to Houghten and colleagues,on Peralkylated Oligopeptide Mixtures discloses linear Cl-C7-alkylperalkylated oligopeptides and sets and libraries of such peptides, aswell as methods for using such oligopeptide sets and libraries fordetermining the sequence of a peralkylated oligopeptide thatpreferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods.

[0214] As one of skill in the art will appreciate, TNF-gamma-alphaand/or TNF-gamma-beta polypeptides of the present invention and theepitope-bearing fragments thereof described above can be combined withparts of the constant domain of immunoglobulins (IgG), resulting inchimeric polypeptides. These fusion proteins facilitate purification andshow an increased half-life in vivo. This has been shown, e.g., forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins (EP A 394,827; Traunecker,et al., Nature 331:84-86 (1988)). Fusion proteins that have adisulfide-linked dimeric structure due to the IgG part can also be moreefficient in binding and neutralizing other molecules than the monomericTNF-gamma protein or protein fragment alone (Fountoulakis, et al., J.Biochem. 270:3958-3964 (1995)). As an example, one such TNF-gamma-Fcfusion has been produced herein as described above.

[0215] Fragments (i.e., portions) of the TNF-gamma polypeptides of thepresent on have uses which include, but are not limited to,intermediates for producing full-length polypeptides.

[0216] For many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron and colleagues (J. Biol. Chem.,268:2984-2988 (1993)) reported modified KGF proteins that had heparinbinding activity even if 3, 8, or 27 N-terminal amino acid residues weremissing. Further, several investigators have reported TNF-alpha muteinsin which two, four or seven N-terminal amino acids had been removedwhich showed a 2- to 3-fold increase in functional activity whencompared to the naturally-occurring TNF-alpha polypeptide (Creasey, A.A., et al., Cancer Res. 47:145-149 (1987); Sidhu, R. S. and Bollon, A.P. Anticancer Res. 9:1569-1576 (1989); Kamijo, R., et al., Biochem.Biophys. Res. Comm. 160:820-827 (1989)). Further, even if deletion ofone or more amino acids from the N-terminus or C-terminus of a proteinresults in modification or loss of one or more biological functions ofthe protein, other TNF-gamma functional activities may still be retained

[0217] In the present case, since the proteins of the invention aremembers of the TNF polypeptide family, deletions of N-terminal aminoacids up to the leucine residue at position 35 of SEQ ID NO:2 (whichcorresponds exactly to the leucine residue at position 134 of SEQ IDNO:20) may retain some biological activity such as regulation of growthand differentiation of many types of hematopoietic and endothelialcells. Polypeptides having further N-terminal deletions including theleucine-36 residue in SEQ ID NO:2 (corresponding to leucine-135 in SEQID NO:20) would not be expected to retain such biological activitiesbecause it is known that this residue in TNF-related polypeptides is inthe beginning of the conserved domain required for biologicalactivities.

[0218] However, even if deletion of one or more amino acids from theN-terminus of a full-length TNF-gamma polypeptide results inmodification or loss of one or more biological functions of thepolypeptide, other biological activities may still be retained. Thus,the ability of the shortened polypeptide to induce and/or bind toantibodies which recognize the full-length or mature form of thepolypeptide generally will be retained when less than the majority ofthe residues of the full-length or mature polypeptide are removed fromthe N-terminus. Whether a particular polypeptide lacking N-terminalresidues of a complete polypeptide retains such immunologic activitiescan readily be determined by routine methods described herein andotherwise known in the art.

[0219] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of the aminoacid sequence of the TNF-gamma-alpha shown in SEQ ID NO:2, up to theleucine residue at position number 35, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n¹-149 of SEQ ID NO:2,where n¹ is an integer in the range of −27 to 35, and 35 is the positionof the first residue from the N-terminus of the complete TNF-gammapolypeptide (shown in SEQ ID NO:2) believed to be required forregulation of growth and differentiation of many types of hematopoieticand endothelial cells.

[0220] In specific embodiments, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively, consisting of, amember selected from the group consisting of the amino acid sequence ofresidues: −27 to 147, −26 to 147, −25 to 147, −24 to 147, −23 to 147,−22 to 147, −21 to 147, −20 to 147, −19 to 147, −18 to 147, −17 to 147,−16 to 147, −15 to 147, −14 to 147, −13 to 147, −12 to 147, −11 to 147,−10 to 147, −9 to 147, −8 to 147, −7 to 147, −6 to 147, −5 to 147, −4 to147, −3 to 147, −2 to 147, −1 to 147, 1 to 147, 2 to 147, 3 to 147, 4 to147, 5 to 147, 6 to 147, 7 to 147, 8 to 147, 9 to 147, 10 to 147, 11 to147, 12 to 147, 13 to 147, 14 to 147, 15 to 147, 16 to 147, 17 to 147,18 to 147, 19 to 147, 20 to 147, 21 to 147, 22 to 147, 23 to 147, 24 to147, 27 to 147, 26 to 147, 27 to 147, 28 to 147, 29 to 147, 30 to 147,31 to 147, 32 to 147, 33 to 147, 34 to 147, and 35 to 14 of SEQ ID NO:2.Polypeptides encoded by these polynucleotides are also encompassed bythe invention. The present invention is also directed to nucleic acidmolecules comprising, or alternatively, consisting of, a polynucleotidesequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%identical to the polynucleotide sequences encoding the TNF-gammapolypeptides described above, and the polypeptides encoded thereby. Thepresent invention also encompasses the above polynucleotide sequencesfused to a heterologous polynucleotide sequence, and the polypeptidesencoded thereby.

[0221] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of the aminoacid sequence of the TNF-gamma-beta shown in SEQ ID NO:20, up to theleucine residue at position number 134, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising the amino acid sequence of residues n²-251 ofSEQ ID NO:20, where n² is an integer in the range of 1 to 134, and 135is the position of the first residue from the N-terminus of the completeTNF-gamma-beta polypeptide (shown in SEQ ID NO:20) believed to berequired for regulation of growth and differentiation of many types ofhematopoietic and endothelial cells activity of the TNF-gamma-betapolypeptide.

[0222] In specific embodiments, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively, consisting of, amember selected from the group consisting of the amino acid sequence ofresidues: 1 to 251, 2 to 251, 3 to 251, 4 to 251, 5 to 251, 6 to 251, 7to 251, 8 to 251, 9to 251, 10 to 251, 11 to 251, 12 to 251, 13 to 251,14 to 251, 15 to 251, 16 to 251, 17 to 251, 18 to 251, 19 to 251, 20 to251, 21 to 251, 22 to 251, 23 to 251, 24 to 251, 25 to 251, 26 to 251,27 to 251, 28 to 251, 29 to 251, 30 to 251, 31 to 251, 32 to 251, 33 to251, 34 to 251, 35 to 251, 36 to 251, 37 to 251, 38 to 251, 39 to 251,40 to 251, 41 to 251, 41 to 251, 42 to 251, 43 to 251, 44 to 251, 45 to251, 46 to 251, 47 to 251, 48 to 251, 49 to 251, 50 to 251, 51 to 251,52 to 251, 53 to 251, 54 to 251, 55 to 251, 56 to 251, 57 to 251, 58 to251, 59 to 251, 60 to 251, 61 to 251, 62 to 251, 63 to 251, 64 to 251,65 to 251, 66 to 251, 67 to 251, 68 to 251, 69 to 251, 70 to 251, 71 to251, 72 to 251, 73 to 251, 74 to 251, 75 to 251, 76 to 251, 77 to 251,78 to 251, 79 to 251, 80 to 251, 81 to 251, 82 to 251, 83 to 251, 84 to251, 85 to 251, 86 to 251, 87 to 251, 88 to 251, 89 to 251, 90 to 251,91 to 251, 92 to 251, 93 to 251, 94 to 251, 95 to 251, 96 to 251, 97 to251, 98 to 251, 99 to 251, 100 to 251, 101 to 251, 102 to 251, 103 to251, 104 to 251, 105 to 251, 106 to 251, 107 to 251, 108 to 251, 109 to251, 110 to 251, 111 to 251, 112 to 251, 113 to 251, 114 to 251, 115 to251, 116 to 251, 117 to 251, 118 to 251, 119 to 251, 120 to 251, 121 to251, 122 to 251, 123 to 251, 124 to 251, 125 to 251, 126 to 251, 127 to251, 128 to 251, 129 to 251, 130 to 251, 131 to 251, 133 to 251, 134 to251, and 134 to 251 of SEQ ID NO:20. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. The presentinvention is also directed to nucleic acid molecules comprising, oralternatively, consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to thepolynucleotide sequences encoding the TNF-gamma polypeptides describedabove, and the polypeptides encoded thereby. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence, and the polypeptides encoded thereby.

[0223] As mentioned above, even if deletion of one or more amino acidsfrom the N-terminus of a polypeptide results in modification of loss ofone or more biological functions of the polypeptide, other biologicalactivities may still be retained. Thus, the ability of the shortenedTNF-gamma-alpha mutein to induce and/or bind to antibodies whichrecognize the full-length or mature form of the polypeptide generallywill be retained when less than the majority of the residues of thefull-length or mature polypeptide are removed from the N-terminus.Whether a particular polypeptide lacking N-terminal residues of acomplete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a TNF-gamma-alpha mutein with a largenumber of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six TNF-gamma-alpha amino acid residues may often evoke an immuneresponse.

[0224] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of thepredicted mature amino acid sequence of the TNF-gamma-alpha shown inFIGS. 1A and 1B (SEQ ID NO:2), up to the phenylalanine residue atposition number 169 of the sequence shown in FIGS. 1A and 1B (whichcorresponds to position number 142 of SEQ ID NO:2) and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides comprising the amino acid sequence of residuesn³-174 of the sequence shown in FIGS. 1A and 1B (n³-147 of SEQ ID NO:2),where n³ is an integer in the range of 1 to 169, and 170 is the positionof the first residue from the N-terminus of the complete TNF-gamma-alphapolypeptide believed to be required for at least immunogenic activity ofthe TNF-gamma-alpha polypeptide.

[0225] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, amember selected from the group consisting of the amino acid sequence ofresidues of R-2 to L-174; R-3 to L-174; F-4 to L-174; L-5 to L-174; S-6to L-174; K-7 to L-174; V-8 to L-174; Y-9 to L-174; L-174; F-11 toL-174; P-12 to L-174; M-13 to L-174; R-14 to L-174; K-15 to L-174; L-16to L-174; I-17 to L-174; L-18 to L-174; F-19 to L-174; L-20 to L-174;V-21 to L-174; F-22 to L-174; P-23 to L-174; V-24 to L-174; V-25 toL-174; R-26 to L-174; Q-27 to L-174; T-28 to L-174; P-29 to L-174; T-30to L-174; Q-31 to L-174; H-32 to L-174; F-33 to L-174; K-34 to L-174;N-35 to L-174; Q-36 to L-174; F-37 to L-174; P-38 to L-174; A-39 toL-174; L-40 to L-174; H-41 to L-174; W-42 to L-174; E-43 to L-174; H-44to L-174; E-45 to L-174; L-46 to L-174; G-47 to L-174; L-48 to L-174;A-49 to L-174; F-50 to L-174; T-51 to L-174; K-52 to L-174; N-53 toL-174; R-54 to L-174; M-55 to L-174; N-56 to L-174; Y-57 to L-174; T-58to L-174; N-59 to L-174; K-60 to L-174; F-61 to L-174; L-62 to L-174;L-63 to L-174; I-64 to L-174; P-65 to L-174; E-66 to L-174; S-67 toL-174; G-68 to L-174; D-69 to L-174; Y-70 to L-174; F-71 to L-174; 1-72to L-174; Y-73 to L-174; S-74 to L-174; Q-75 to L-174; V-76 to L-174;T-77 to L-174; F-78 to L-174; R-79 to L-174; G-80 to L-174; M-81 toL-174; T-82 to L-174; S-83 to L-174; E-84 to L-174; C-85 to L-174; S-86to L-174; E-87 to L-174; 1-88 to L-174; R-89 to L-174; Q-90 to L-174;A-91 to L-174; G-92 to L-174; R-93 to L-174; P-94 to L-174 ; N-95 toL-174; K-96 to L-174; P-97 to L-174; D-98 to L-174; S-99 to L-174; I-100to L-174; T-101 to L-174; V-102 to L-174; V-103 to L-174; I-104 toL-174; T-105 to L-174; K-106 to L-174; V-107 to L-174; T-108 to L-174;D-109 to L-174; S-110 to L-174; Y-111 to L-174; P-1 12 to L-174; E-113to L-174; P-114 to L-174; T-115 to L-174; Q-116 to L-174; L-117 toL-174; L-118 to L-174; M-119 to L-174; G-120 to L-174; T-121 to L-174;K-122 to L-174; S-123 to L-174; V-124 to L-174; C-125 to L-174; E-126 toL-174; V-127 to L-174; G-128 to L-174; S-129 to L-174; N-130 to L-174;W-131 to L-174; F-132 to L-174; Q-133 to L-174; P-134 to L-174; I-135 toL-174; Y-136 to L-174; L-137 to L-174; G-138 to L-174; A-139 to L-174;M-140 to L-174; F-141 to L-174; S-142 to L-174; L-143 to L-174; Q-144 toL-174; E-145 to L-174; G-146 to L-174; D-147 to L-174; K-148 to L-174;L-149 to L-174; M-150 to L-174; V-151 to L-174; N-152 to L-174; V-153 toL-174; S-154 to L-174; D-155 to L-174; I-156 to L-174; S-157 to L-174;L-158 to L-174; V-159 to L-174; D-160 to L-174; Y-161 to L-174; T-162 toL-174; K-163 to L-174; E-164 to L-174; D-165 to L-174; K-166 to L-174;T-167 to L-174; F-168 to L-174; and F-169 to L-174 of theTNF-gamma-alpha sequence shown in FIGS. 1A and 1B (the TNF-gamma-alphaamino acid sequence shown in FIGS. 1A and 1B is identical to that in SEQID NO:2, however, the numbering scheme differs between the two; thenumbering of the above amino acid residues in this case reflects that ofFIGS. 1A and 1B). Polypeptides encoded by these polynucleotides are alsoencompassed by the invention. The present invention is also directed tonucleic acid molecules comprising, or alternatively, consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%,98% or 99% identical to the polynucleotide sequences encoding theTNF-gamma polypeptides described above, and the polypeptides encodedthereby. The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence, and thepolypeptides encoded thereby.

[0226] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of thepredicted mature amino acid sequence of the TNF-gamma-beta shown in SEQID NO:20, up to the phenylalanine residue at position number 246 andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising the amino acid sequence ofresidues n⁴-251 of SEQ ID NO:20, where n⁴ is an integer in the range of2 to 246, and 247 is the position of the first residue from theN-terminus of the complete TNF-gamma-beta polypeptide believed to berequired for at least immunogenic activity of the TNF-gamma-betaprotein.

[0227] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, amember selected from the group consisting of the amino acid sequence ofresidues of A-2 to L-251; E-3 to L-251; D-4 to L-251; L-5 to L-251; G-6to L-251; L-7 to L-251; S-8 to L-251; F-9 to L-251; G-10 to L-251; E-11to L-251; T-12 to L-251; A-13 to L-251; S-14 to L-251; V-15 to L-251E-16 to L-251; M-17 to L-251; L-18 to L-251; P-19 to L-251; E-20 toL-251; H-21 to L-251; G-22 to L-251; S-23 to L-251; C-24 to L-251; R-25to L-251; P-26 to L-251; K-27 to L-251; A-28 to L-251; R-29 to L-251;S-30 to L-251; S-31 to L-251; S-32 to L-251; A-33 to L-251; R-34 toL-251; W-35 to L-251; A-36 to L-251; L-37 to L-251; T-38 to L-251; C-39to L-251; C-40 to L-251; L-41 to L-251; V-42 to L-251; L-43 to L-251;L-44 to L-251; P-45 to L-251; F-46 to L-251; L-47 to L-251; A-48 toL-251; G-49 to L-251; L-50 to L-251; T-51 to L-251; T-52 to L-251; Y-53to L-251; L-54 to L-251; L-55 to L-251; V-56 to L-251; S-57 to L-251;Q-58 to L-251; L-59 to L-251; R-60 to L-251; A-61 to L-25 1; Q-62 toL-25 1; G-63 to L-251; E-64 to L-251; A-65 to L-251; C-66 to L-251; V-67to L-251; Q-68 to L-251; F-69 to L-251; Q-70 to L-251; A-71 to L-251;L-72 to L-251; K-73 to L-251; G-74 to L-251; Q-75 to L-251; E-76 toL-251; F-77 to L-251; A-78 to L-251; P-79 to L-251; S-80 to L-251; H-81to L-251; Q-82 to L-251; Q-83 to L-251; V-84 to L-251; Y-85 to L-251;A-86 to L-251; P-87 to L-251; L-88 to L-251; R-89 to L-251; A-90 to L-251; D-91 to L-25 1; G-92 to L-25 1; D-93 to L-25 1; K-94 to L-25 1; P-95to L-251; R-96 to L-251; A-97 to L-251; H-98 to L-251; L-99 to L-251;T-100 to L-251; V-101 to L-251; V-102 to L-251; R-103 to L-251; Q-104 toL-251; T-105 to L-251; P-106 to L-251; T-107 to L-251; Q-108 to L-251;H-109 to L-251; F-110 to L-251; K-111 to L-251; N-112 to L-251; Q-113 toL-251; F-114 to L-251; P-115 to L-251; A-116 to L-251; L-117 to L-251;H-118 to L-251; W-119 to L-251; E-120 to L-251; H-121 to L-251; E-122 toL-251; L-123 to L-251; G-124 to L-251; L-125 to L-251; A-126 to L-251;F-127 to L-251; T-128 to L-251; K-129 to L-251; N-130 to L-251; R-131 toL-251; M-132 to L-251; N-133 to L-251; Y-134 to L-251; T-135 to L-251;N-136 to L-251; K-137 to L-251; F-133 to L-251; L-139 to L-251; L-140 toL-251; N-141 to L-251; P-142 to L-251; E-143 to L-251; S-144 to L-251;G-145 to L-251; D-146 to L-251; Y-147 to L-251; F-148 to L-251; I-149 toL-251; Y-150 to L-251; S-151 to L-251; Q-152 to L-251; V-153 to L-251;T-154 to L-251; F-155 to L-251; R-156 to L-251; G-157 to L-25 1; M-158to L-251; T-159 to L-251; S-160 to L-251; E-161 to L-251; C-162 L-251;S-163 to L-251; E-164 to L-251; I-165 to L-251; R-166 to L-251; Q-167 toL-251; A-168 to L-251; G-169 to L-251; R-170 to L-251; P-171 to L-251;N-172 to L-251; K-173 to L-251; P-174 to L-251; D-175 to L-251; S-176 toL-251; I-177 to L-251; T-178 to L-251; V-179 to L-251; V-180 to L-251;I-181 to L-251; T-182 to L-251; V-184 to L-251; T-185 to L-251; D-186 toL-251; S-187 to L-251; Y-188 to L-251; P-189 to L-251; E-190 to L-251;P-191 to L-251; T-192 to L-251; Q-193 to L-251; L-194 to L-251; L-195 toL-251; M-196 to L-251; G-197 to L-251; T-198 to L-251; K-199 to L-251;S-200 to L-251; V-201 to L-251; C-202 to L-251; E-203 to L-251; V-204 toL-251; G-205 to L-251; S-206 to L-251; N-207 to L-251; W-208 to L-251;F-209 to L-251; Q-210 to L-251; P-211 to L-251; I-212 to L-251; Y-213 toL-251; L-214 to L-251; G-215 to L-251; A-216 to L-251; M-217 to L-251;F-218 to L-251; S-219 to L-251; L-220 to L-251; Q-221 to L-251; E-222 toL-251; G-223 to L-251; D-224 to L-251; K-225 to L-251; L-226 to L-251;M-227 to L-251; V-228 to L-251; N-229 to L-251; V-230 to L-251; S-231 toL-251; D-232 to L-251; I-233 to L-251; S-234 to L-251; L-235 to L-251;V-236 to L-251; D-237 to L-251; Y-238 to L-251; T-239 to L-251; K-240 toL-251; E-241 to L-251; D-242 to L-251; K-243 to L-251; T-244 to L-251;F-245 to L-251; and F-246 to L-251 of the TNF-gamma-beta sequence shownin SEQ ID NO:20. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention. The present invention is also directed tonucleic acid molecules comprising, or alternatively, consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%,98% or 99% identical to the polynucleotide sequences encoding theTNF-gamma polypeptides described above, and the polypeptides encodedthereby. The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence, and thepolypeptides encoded thereby.

[0228] Similarly, many examples of biologically functional C-terminaldeletion muteins are known. For instance, Interferon gamma shows up toten times higher activities by deleting 8 to 10 amino acid residues fromthe carboxy terminus of the protein (Dobeli, et al., J. Biotechnology7:199-216 (1988)). Further, several investigators have reportedbiologically inactive TNF-alpha muteins in which as few as two aminoacids had been removed from the C-terminus (Carlino, J. A., et al., J.Biol. Chem. 262:958-961 (1987); Creasey, A. A., et al., Cancer Res.47:145-149 (1987); Sidhu, R. S. and Bollon, A. P. Anticancer Res.9:1569-1576 (1989); Gase, K., et al., Immunology 71:368-371 (1990)).

[0229] In the present case, since the proteins of the invention aremembers of the TNF polypeptide family, deletions of C-terminal aminoacids up to the leucine at position 146 of SEQ ID NO:2 (whichcorresponds to the leucine at position 250 of SEQ ID NO:20) may retainsome biological activity such as regulation of growth anddifferentiation of many types of hematopoietic and endothelial cells.Polypeptides having further C-terminal deletions including the leucineresidue at position 146 of SEQ ID NO:2 (or the leucine residue atposition 250 of SEQ ID NO:20) would not be expected to retain suchbiological activities because it is known that this residue inTNF-related polypeptides is in the beginning of the conserved domainrequired for biological activities.

[0230] However, even if deletion of one or more amino acids from theC-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of the shortened protein to induceand/or bind to antibodies which recognize the complete or mature form ofthe protein generally will be retained when less than the majority ofthe residues of the complete or mature protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

[0231] In additional embodiments, the present invention further providespolypeptides having one or more residues removed from the carboxyterminus of the amino acid sequence of the TNF-gamma-alpha shown in SEQID NO:2, up to the leucine residue at position 146 of SEQ ID NO:2, andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides having the amino acid sequence ofresidues −27-m¹ of the amino acid sequence in SEQ ID NO:2, where m¹ isany integer in the range of 146 to 147, and residue 146 is the positionof the first residue from the C-terminus of the complete TNF-gamma-alphapolypeptide (shown in SEQ ID NO:2) believed to be required forregulation of growth and differentiation of many types of hematopoieticand endothelial cells by the TNF-gamma-alpha polypeptide.

[0232] More in particular, the invention provides polynucleotidesencoding polypeptides having the amino acid sequence of residues −27-146and −27-147 of SEQ ID NO:2. Polynucleotides encoding these polypeptidesalso are provided.

[0233] The present invention also provides polypeptides having one ormore residues removed from the carboxy terminus of the amino acidsequence of the TNF-gamma-beta shown in SEQ ID NO:20, up to the leucineresidue at position 250 of SEQ ID NO:20, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides having the amino acid sequence of residues 1-m² of theamino acid sequence in SEQ ID NO:20, where m² is any integer in therange of 250 to 251, and residue 249 is the position of the firstresidue from the C- terminus of the complete TNF-gamma-beta polypeptide(shown in SEQ ID NO:20) believed to be required for regulation of growthand differentiation of many types of hematopoietic and endothelialcells.

[0234] More in particular, the invention provides polynucleotidesencoding polypeptides having the amino acid sequence of residues 1-250and 1-251 of SEQ ID NO:20. Polynucleotides encoding these polypeptidesalso are provided.

[0235] The invention also provides polypeptide fragments comprising, oralternatively consisting of, one or more amino acids deleted from boththe amino and the carboxyl termini of TNF-gamma-alpha, which may bedescribed generally as having residues n¹-m¹ of SEQ ID NO:2, where n andm are integers as described above. The invention further providespolypeptides having one or more amino acids deleted from both the aminoand the carboxyl termini of TNF-gamma-beta, which may be describedgenerally as having residues n²-m² of SEQ ID NO:20, where n² and m² areintegers as described above.

[0236] As mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a polypeptide results in modification of loss ofone or more biological functions of the polypeptide, other biologicalactivities may still be retained. Thus, the ability of the shortenedTNF-gamma-alpha mutein to induce and/or bind to antibodies whichrecognize the full-length or mature of the polypeptide generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a full-length polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a TNF-gamma-alpha mutein with a large number of deletedC-terminal amino acid residues may retain some biological or immunogenicactivities. In fact, peptides composed of as few as six TNF-gamma-alphaamino acid residues may often evoke an immune response.

[0237] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the TNF-gamma-alpha shown in FIGS. 1A and 1B (orin SEQ ID NO:2), up to the serine residue at position number 6 in FIGS.1A and 1B (or −22 in SEQ ID NO:2), and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m³ of SEQ ID NO:2,where m³ is an integer in the range of 6 to 174, and 6 is the positionof the first residue from the C-terminus of the complete TNF-gamma-alphapolypeptide believed to be required for at least immunogenic activity ofTNF-gamma-alpha.

[0238] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, amember selected from the group consisting of the amino acid sequence ofresidues M-1 to L-173; M-1 to F-172; M-1 to A-171; M-1 to G-170; M-1 toF-169; M-1 to F-168; M-1 to T-167; M-1 to K-166; M-1 to D-165; M-1 toE-164; M-1 to K-163; M-1 to T-162; M-1 to Y-161; M-1 to D-160; M-1 toV-159; M-1 to L-158; M-1 to S-157; M-1 to I-156; M-1 to D-155; M-1 toS-154; M-1 V-153; M-1 to N-152; M-1 to V-151; M-1 to M-150; M-1 toL-149; M-1 to K-148; M-1 to D-147; M-1 to G-146; M-1 to E-145; M-1 toQ-144; M-1 to L-143; M-1 to S-142; M-1 to F-141; M-1 to M-140; M-1 toA-139; M-1 to G-138; M-1 to L-137; M-1 to Y-136; M-1 to I-135; M-1 toP-134; M-1 to Q-133; M-1 to F-132; M-1 to W-131; M-1 to N-130; M-1 toS-129; M-1 to G-128; M-1 to V-127; M-1 to E-126; M-1 to C-125; M-1 toV-124; M-1 to S-123; M-1 to K-122; M-1 to T-121; M-1 to G-120; M-1 toM-119; M-1 to L-118; M-1 to L-117; M-1 to Q-116; M-1 to T-115; M-1 toP-114; M-1 to E-113; M-1 to P-112; M-1 to Y-111; M-1 to S-110; M-1 toD-109; M-1 to T-108; M-1 to V-107; M-1 to K-106; M-1 to T-105; M-1 toI-104; M-1 to V-103; M-1 to V-102; M-1 to T-101; M-1 to I-100; M-1 toS-99; M-1 to D-98; M-1 to P-97; M-1 to K-96; M-1 to N-95; M-1 to P-94;M-1 to R-93; M-1 to G-92; M-1 to A-91; M-1 to Q-90; M-1 to R-89; M-1 toI-88; M-1 to E-87; M-1 to S-86; M-1 to C-85; M-1 to E-84; M-1 to S-83;M-1 to T-82; M-1 to M-81; M-1 to G-80; M-1 to R-79; M-1 to F-78; M-1 toT-77; M-1 to V-76; M-1 to Q-75; M-1 to S-74; M-1 to Y-73; M-1 to I-72;M-1 to F-71; M-1 to Y-70; M-1 to D-69; M-1 to G-68; M-1 to S-67; M-1 toE-66; M-1 to P-65; M-1 to I-64; M-1 to L-63; M-1 to L-62; M-1 to F-61;M-1 to K-60; M-1 to N-59; M-1 to T-58; M-1 to Y-57; M-1 to N-56; M-1 toM-55; M-1 to R-54; M-1 to N-53; M-1 to K-52; M-1 to T-51; M-1 to F-50;M-1 to A-49; M-1 to L-48; M-1 to G-47; M-1 to L-46; M-1 to E-45; M-1 toH-44; M-1 to E-43; M-1 to W-42; M-1 to H-41; M-1 to L-40; M-1 to A-39;M-1 to P-38; M-1 to F-37; M-1 to Q-36; M-1 to N-35; M-1 to K-34; M-1 toF-33; M-1 to H-32; M-1 to Q-31; M-1 to T-30; M-1 to P-29; M-1 to T-28;M-1 to Q-27; M-1 to R-26; M-1 to V-25; M-1 to V-24; M-1 to P-23; M-1 toF-22; M-1 to V-21; M-1 to L-20; M-1 to F-19; M-1 to L-18; M-1 to I-17;M-1 to L-16; M-1 to K-15; M-1 to R-14; M-1 to M-13; M-1 to P-12; M-1 toF-11; M-1 to S-10; M-1 to Y-9; M-1 to V-8; M-1 to K-7; and M-1 to S-6 ofthe sequence of the TNF-gamma-alpha sequence shown in FIGS. 1A and 1B(the TNF-gamma-alpha amino acid sequence shown in FIGS. 1A and 1B isidentical to that in SEQ ID NO:2, however, the numbering scheme differsbetween the two; the numbering of the above amino acid residues in thiscase reflects that of FIGS. 1A and 1B). Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. The presentinvention is also directed to nucleic acid molecules comprising, oralternatively, consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to thepolynucleotide sequences encoding the TNF-gamma polypeptides describedabove, and the polypeptides encoded thereby. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence, and the polypeptides encoded thereby.

[0239] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini of aTNF-gamma-alpha polypeptide, which may be described generally as havingresidues n³-m³ of SEQ ID NO:2, where n³ and m³ are integers as describedabove. Polynucleotides encoding the polypeptides are also encompassed bythe invention.

[0240] The present invention further provides polypeptides having one ormore residues deleted from the carboxy terminus of the amino acidsequence of the TNF-gamma-beta shown in SEQ ID NO:20, up to the glycineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m⁴ of SEQ ID NO:20,where m⁴ is an integer in the range of 6 to 250, and 6 is the positionof the first residue from the C-terminus of the complete TNF-gamma-betapolypeptide believed to be required for at least immunogenic activity ofthe TNF-gamma-beta protein.

[0241] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, amember selected from the group consisting of the amino acid sequence ofresidues M-1 to L-250; M-1 to F-249; M-1 to A-248; M-1 to G-247; M-1 toF-246; M-1 to F-245; M-1 T-244; to K-243; M-1 to D-242; M-1 to E-241;M-1 to K-240; M-1 to T-239; M-1 to Y-238; M-1 to D-237; M-1 to V-236;M-1 to L-235; M-1 to S-234; M-1 to I-233; M-1 to D-232; M-1 to S-23 M-1to V-230; M-1 to N-229; M-1 to V-228; M-1 to M-227; M-1 to L-226; M-1 toK-225; M-1 to D-224; M-1 to G-223; M-1 to E-222; M-1 to Q-221; M-1 toL-220; M-1 to S-219; M-1 to F-218; M-1 to M-217; M-1 to A-216; M-1 toG-215; M-1 to L-214; M-1 to Y-213; M-1 to I-212; M-1 to P-211; M-1 toQ-210; M-1 to F-209; M-1 to W-208; M-1 to N-207; M-1 to S-206; M-1 toG-205; M-1 to V-204; M-1 to E-203; M-1 to C-202; M-1 to V-201; M-1 toS-200; M-1 to K-199; M-1 to T-198; M-1 to G-197; M-1 to M-196; M-1 toL-195; M-1 to L-194; M-1 to Q-193; M-1 to T-192; M-1 to P-191; M-1 toE-190; M-1 to P-189; M-1 to Y-188; M-1 to S-187; M-1 to D-186; M-1 toT-185; M-1 to V-184; M-1 to K-183; M-1 to T-182; M-1 to I-181; M-1 toV-180; M-1 to V-179; M-1 to T-178; M-1 to I-177; M-1 to S-176; M-1 toD-175; M-1 to P-174; M-1 to K-173; M-1 to N-172; M-1 to P-171; M-1 toR-170; M-1 to G-169; M-1 to A-168; M-1 to Q-167; M-1 to R-166; M-1 toI-165; M-1 to E-164; M-1 to S-163; M-1 to C-162; M-1 to E-161; M-1 toS-160; M-1 to T-159; M-1 to M-158; M-1 to G-157; M-1 to R-156; M-1 toF-155; M-1 to T-154; M-1 to V-153; M-1 to Q-152; M-1 to S-151; M-1 toY-150; M-1 to I-149; M-1 to F-148; M-1 to Y-147; M-1 to D-146; M-1 toG-145; M-1 to S-144; M-1 to E-143; M-1 to P-142; M-1 to I-141; M-1 toL-140; M-1 to L-139; M-1 to F-138; M-1 to K-137; M-1 to N-136; M-1 toT-135; M-1 to Y-134; M-1 to N-133; M-1 to M-132; M-1 to R-131; M-1 toN-130; M-1 to K-129; M-1 to T-128; M-1 to F-127; M-1 to A-126; M-1 toL-125; M-1 to G-124; M-1 to L-123; M-1 to E-122; M-1 to H-121; M-1 toE-120; M-1 to W-119; M-1 to H-118; M-1 to L-117; M-1 to A-116; M-1 toP-115; M-1 to F-114; M-1 to Q-113; M-1 to N-112; M-1 to K-111; M-1 toF110; M-1 to H-109; M-1 to Q-108; M-1 to T-107; M-1 to P-106; M-1 toT-105; M-1 to Q-104; M-1 to R-103; M-1 to V-102; M-1 to V-101; M-1 toT-100; M-1 to L-99; M-1 to H-98; M-1 to A-97; M-1 to R-96; M-1 to P-95;M-1 to K-94; M-1 to D-93; M-1 to G-92; M-1 to D-91; M-1 to A-90; M-1 toR-89; M-1 to L-88; M-1 to P-87; M-1 to A-86; M-1 to Y-85; M-1 to V-84;M-1 to Q-83; M-1 to Q-82; M-1 to H-81; M-1 to S-80; M-1 to P-79; M-1 toA-78; M-1 to F-77; M-1 to E-76; M-1 to Q-75; M-1 to G-74; M-1 to K-73;M-1 to L-72; M-1 to A-71; M-1 to Q-70; M-1 to F-69; M-1 to Q-68; M-1 toV-67; M-1 to C-66; M-1 to A-65; M-1 to E-64; M-1 to G-63; M-1 to Q-62;M-1 to A-61; M-1 to R-60; M-1 to L-59; M-1 to Q-58; M-1 to S-57; M-1 toV-56; M-1 to L-55; M-1 to L-54; M-1 to Y-53; M-1 to T-52; M-1 to T-51;M-1 to L-50; M-1 to G-49; M-1 to A-48; M-1 to L-47; M-1 to F-46; M-1 toP-45; M-1 to L-44; M-1 to L-43; M-1 to V-42; M-1 to L-41; M-1 to C-40;M-1 to C-39; M-1 to T-38; M-1 to L-37; M-1 to A-36; M-1 to W-35; M-1 toR-34; M-1 to A-33; M-1 to S-32; M-1 to S-31; M-1 to S-30; M-1 to R-29;M-1 to A-28; M-1 to K-27; M-1 to P-26; M-1 to R-25; M-1 to C-24; M-1 toS-23; M-1 to G-22; M-1 to H-21; M-1 to E-20; M-1 to P-19; M-1 to L-18;M-1 to M-17; M-1 to E-16; M-1 to V-15; M-1 to S-14; M-1 to A-13; M-1 toT-12; M-1 to E-11; M-1 to G-10; M-1 to F-9; M-1 to S-8; M-1 to L-7; andM-1 to G-6 of the sequence of the TNF-gamma-beta sequence shown in SEQID NO:20. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention. The present invention is also directed tonucleic acid molecules comprising, or alternatively, consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%,98% or 99% identical to the polynucleotide sequences encoding theTNF-gamma polypeptides described above, and the polypeptides encodedthereby. The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence, and thepolypeptides encoded thereby.

[0242] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini of aTNF-gamma-beta polypeptide, which may be described generally as havingresidues n⁴-m⁴ of SEQ ID NO:20, where n⁴ and m⁴ are integers asdescribed above. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0243] Further embodiments of the invention are directed to polypeptidefragments comprising, or alternatively, consisting of, amino acidsdescribed by the general formula m^(x) to n^(x), where m and ncorrespond to any one of the amino acid residues specified above forthese symbols, respectively, and x represents any integer.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0244] In additional embodiments, the present invention providespolynucleotides encoding polypeptides comprising the amino acid sequenceof residues 72-m⁴ of FIG. 1 (i.e., SEQ ID NO:2), where m⁴ is an integerfrom 78 to 250, corresponding to the position of the amino acid residuein SEQ ID NO:20. For example, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, amember selected from the group consisting of the amino acid sequence ofresidues L-72 to L-250; L-72 to F-249; L-72 to A-248; L-72 to G-247;L-72 to F-246; L-72 to F-245; L-72 to T-244; L-72 to K-243; L-72 toD-242; L-72 to E-241; L-72 to K-240; L-72 to T-239; L-72 to Y-238; L-72to D-237; L-72 to V-236; L-72 to L-235; L-72 to S-234; L-72 to I-233;L-72 to D-232; L-72 to S-231; L-72 to V-230; L-72 to N-229; L-72 toV-228; L-72 to M-227; L-72 to L-226; L-72 to K-225; L-72 to D-224; L-72to G-223; L-72 to E-222; L-72 to Q-221; L-72 to L-220; L-72 to S-219;L-72 to F-218; L-72 to M-217; L-72 to A-216; L-72 to G-215; L-72 toL-214; L-72 to Y-213; L-72 to I-212; L-72 to P-211; L-72 to Q-210; L-72to F-209; L-72 to W-208; L-72 to N-207; L-72 to S-206; L-72 to G-205;L-72 to V-204; L-72 to E-203; L-72 to C-202; L-72 to V-201; L-72 toS-200; L-72 to K-199; L-72 to T-198; L-72 to G-197; L-72 to M-196; L-72to L-195; L-72 to L-194; L-72 to Q-193; L-72 to T-192; L-72 to P-191;L-72 to E-190; L-72 to P-189; L-72 to Y-188; L-72 to S-187; L-72 toD-186; L-72 to T-185; L-72 to V-184; L-72 to K-183; L-72 to T-182; L-72to I-181; L-72 to V-180; L-72 to V-179; L-72 T-178; L-72 to I-177; L-72to S-176; L-72 to D-175; L-72 to P-174; L-72 to K-173; L-72 to N-172;L-72 to P-171; L-72 to R-170; L-72 to G-169; L-72 to A-168; L-72 toQ-167; L-72 to R-166; L-72 to I-165; L-72 to E-164; L-72 to S-163; L-72to C-162; L-72 to E-161; L-72 to S-160; L-72 to T-159; L-72 to M-158;L-72 to G-157; L-72 to R-156; L-72 to F-155; L-72 to T-154; L-72 toV-153; L-72 to Q-152; L-72 to S-151; L-72 to Y-150; L-72 to I-149; L-72to F-148; L-72 to Y-147; L-72 to D-146; L-72 to G-145; L-72 to S-144;L-72 to E-143; L-72 to P-142; L-72 to I-141; L-72 to L-140; L-72 toL-139; L-72 to F-138; L-72 to K-137; L-72 to N-136; L-72 to T-135; L-72to Y-134; L-72 to N-133; L-72 to M-132; L-72 to R-131; L-72 to N-130;L-72 to K-129; L-72 to T-128; L-72 to F-127; L-72 to A-126; L-72 toL-125; L-72 to G-124; L-72 to L-123; L-72 to E-122; L-72 to H-121; L-72to E-120; L-72 to W-119; L-72 to H-1 18; L-72 to L-117; L-72 to A-116;L-72to P-115; L-72to F-114; L-72 to Q-113; L-72 to N-112; L-72 to K-111;L-72 to F-110; L-72 to H-109; L-72 to Q-108; L-72 to T-107; L-72 toP-106; L-72 to T-105; L-72 to Q-104; L-72 to R-103; L-72 to V-102; L-72to V-101; L-72 to T-100; L-72 to L-99; L-72 to H-98; L-72 to A-97; L-72to R-96; L-72 to P-95; L-72 to K-94; L-72 to D-93; L-72 to G-92; L-72 toD-91; L-72 to A-90; L-72 to R-89; L-72 to L-88; L-72 to P-87; L-72 toA-86; L-72 to Y-85; L-72 to V-84; L-72 to Q-83; L-72 to Q-82; L-72 toH-81; L-72 to S-80; L-72 to P-79; L-72 to A-78; of the sequence of theTNF-gamma-beta sequence shown in SEQ ID NO:20. The present applicationis also directed to nucleic acid molecules comprising, or alternatively,consisting of, a polynucleotide sequence at least 85%, 90%, 92%, 94%,95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequencedescribed above. The present invention also encompasses thesepolynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these nucleic acids and/orpolynucleotide sequences are also encompassed by the invention.

[0245] Specific embodiments of the invention are directed to nucleotidesequences encoding a polypeptide consisting of a portion of the completeTNF-gamma-alpha amino acid sequence encoded by the cDNA clone containedin ATCC Deposit No. 75927, where this portion excludes from 1 to about62 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 75927,or about 1 amino acid from the carboxy terminus, or any combination ofthe above amino terminal and carboxy terminal deletions, of the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 75927. Polynucleotides encoding all of the above deletion mutantpolypeptide forms also are provided.

[0246] In another embodiment, the invention is directed to a nucleotidesequence encoding a polypeptide consisting of a portion of the completeTNF-gamma-beta amino acid sequence encoded by the cDNA clone containedin ATCC Deposit No. 203055, where this portion excludes from 1 to about134 amino acids from the amino terminus of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 203055,or excludes a number of amino acids from the amino terminus of thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 203055 (where the number is selected from any integer from 1to 134), or about 1 amino acid from the carboxy terminus, or anycombination of the above amino terminal and carboxy terminal deletions,of the complete amino acid sequence encoded by the cDNA clone containedin ATCC Deposit No. 203055. Polynucleotides encoding all of the abovepolypeptides are also encompassed by the invention.

[0247] The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-alphapolypeptide having the complete amino acid sequence shown in SEQ ID NO:2(i.e., positions −27 to 147 of SEQ ID NO:2); (b) the amino acid sequenceof the full-length TNF-gamma-alpha polypeptide having the complete aminoacid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine(i.e., positions −26 to 147 of SEQ ID NO:2); (c) the amino acid sequenceof the predicted mature TNF-gamma-alpha polypeptide having the aminoacid sequence at positions 1-147 in SEQ ID NO:2 (d) the complete aminoacid sequence encoded by the cDNA clone HUVEO91 contained in the ATCCDeposit No. 75927; (e) the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone contained in the ATCCDeposit No. 75927; and (f) the complete amino acid sequence of thepredicted mature TNF-gamma polypeptide encoded by the cDNA clone HUVEO91contained in the ATCC Deposit No. 75927. The polypeptides of the presentinvention also include polypeptides having an amino acid sequence atleast 70% identical, at least 80% or 85% identical, more preferably atleast 90%, 92% or 94% identical, and still more preferably 95%, 96%,97%, 98% or 99% identical to those described in (a), (b), (c), (d), (e)or (f), above, or fragments thereof, as described herein.

[0248] The invention further provides an isolated TNF-gamma polypeptidecomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of the full-length TNF-gamma-betapolypeptide having the complete amino acid sequence shown in SEQ IDNO:20 (i.e., positions 1 to 251 of SEQ ID NO:20); (b) the amino acidsequence of the full-length TNF-gamma-beta polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:20 excepting theN-terminal methionine (i.e., positions 2 to 251 of SEQ ID NO:20); (c)the amino acid sequence of the predicted mature TNF-gamma-betapolypeptide having the amino acid sequence at positions 62-251 in SEQ IDNO:20; (d) the complete amino acid sequence encoded by the cDNA cloneHEMCZ56 contained in the ATCC Deposit No. 203055; (e) the complete aminoacid sequence excepting the N-terminal methionine encoded by the cDNAclone HEMCZ56 contained in the ATCC Deposit No. 203055; and (f) thecomplete amino acid sequence of the predicted mature TNF-gammapolypeptide encoded by the cDNA clone HEMCZ56 contained in the ATCCDeposit No. 203055. The polypeptides of the present invention alsoinclude polypeptides having an amino acid sequence at least 70%identical, at least 80% or 85% identical, more preferably at least 90%,92% or 94% identical, and still more preferably 95%, 96%, 97%, 98% or99% identical to those described in (a), (b), (c), (d), (e) or (f),above, or fragments thereof, as described herein. In specificembodiments, these polypeptides are at least 10 amino acids, at least 15amino acids, at least 20 amino acids, at least 25 amino acids, at least30 amino acids and more preferably at least 50 amino acids.

[0249] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of aTNF-gamma polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the TNF-gammapolypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

[0250] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIGS. 1A and B (SEQ ID NO:2), the aminoacid sequence encoded by deposited cDNA clone HUVEO91, or fragmentsthereof, can be determined conventionally using known computer programssuch the Bestfit program (Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

[0251] In a specific embodiment, the identity between a reference(query) sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the FASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that are N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. A determination of whether a residue is matched/alignedis determined by results of the FASTDB sequence alignment. Thispercentage is then subtracted from the percent identity, calculated bythe above FASTDB program using the specified parameters, to arrive at afinal percent identity score. This final percent identity score is whatis used for the purposes of this embodiment. Only residues to the N- andC-termini of the subject sequence, which are not matched/aligned withthe query sequence, are considered for the purposes of manuallyadjusting the percent identity score. That is, only query residuepositions outside the farthest N- and C-terminal residues of the subjectsequence. For example, a 90 amino acid residue subject sequence isaligned with a 100 residue query sequence to determine percent identity.The deletion occurs at the N-terminus of the subject sequence andtherefore, the FASTDB alignment does not show a matching/alignment ofthe first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for. No other manualcorrections are made for the purposes of this embodiment.

[0252] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0253] The polypeptides of the present invention also include thepolypeptide of SEQ ID NO:20 (in particular the extracellular domain ofthe polypeptide) as well as polypeptides which have at least 70%similarity (preferably at least 70% identity) to the polypeptide of SEQID NO:20 and more preferably at least 90% similarity (more preferably atleast 90% identity) to the polypeptide of SEQ ID NO:20 and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide of SEQ ID NO:20 and also include portionsof such polypeptides with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids.

[0254] Further polypeptides of the present invention includepolypeptides have at least 70% similarity, at least 90% similarity, morepreferably at least 95% similarity, and still more preferably at least96%, 97%, 98% or 99% similarity to those polypeptides described herein.The polypeptides of the invention also comprise those which are at least70% identical, at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptides disclosed herein. In specific embodiments,such polypeptides comprise at least 30 amino acids and more preferablyat least 50 amino acids.

[0255] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide. By “% similarity” for two polypeptides is intended asimilarity score produced by comparing the amino acid sequences of thetwo polypeptides using the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) and the defaultsettings for determining similarity. Bestfit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics2:482-489, 1981) to find the best segment of similarity between twosequences.

[0256] The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. 75927 or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO:1 or contained in ATCC depositNo. 75927 under stringent hybridization conditions or lower stringencyhybridization conditions as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:1), polynucleotide sequences of thecomplementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to thecomplementary strand under stringent hybridization conditions or lowerstringency hybridization conditions defined supra.

[0257] The present invention also encompasses polypeptides comprising,or alternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:20, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. 203055 or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO:19 or contained in ATCC depositNo. 203055 under stringent hybridization conditions or lower stringencyhybridization conditions as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:19), polynucleotide sequences of thecomplementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to thecomplementary strand under stringent hybridization conditions or lowerstringency hybridization conditions defined supra.

[0258] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0259] Fragments which function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0260] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in immunoassays. (See, for instance, Wilson et al.,Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

[0261] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

[0262] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,66:2347-2354 (1985). If in vivo immunization is used, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine residues may be coupled to a carrier usinga linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),while other peptides may be coupled to carriers using a more generallinking agent such as glutaraldehyde. Animals such as rabbits, rats andmice are immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 μg of peptide or carrier protein and Freund'sadjuvant or any other adjuvant known for stimulating an immune response.Several booster injections may be needed, for instance, at intervals ofabout two weeks, to provide a useful titer of anti-peptide antibodywhich can be detected, for example, by ELISA assay using free peptideadsorbed to a solid surface. The titer of anti-peptide antibodies inserum from an immunized animal may be increased by selection ofanti-peptide antibodies, for instance, by adsorption to the peptide on asolid support and elution of the selected antibodies according tomethods well known in the art.

[0263] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublications WO 96/22024 and WO 99/04813). IgG Fusion proteins that havea disulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

[0264] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. In another embodiment, alteration ofpolynucleotides corresponding to SEQ ID NO:19 and the polypeptidesencoded by these polynucleotides may be achieved by DNA shuffling. Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:25 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

[0265] The TNF-gamma-alpha and TNF-gamma-beta polypeptides (proteins) ofthe invention may be in monomers or multimers (i.e., dimers, trimers,tetramers and higher multimers). In specific embodiments, thepolypeptides of the invention are monomers, dimers, trimers ortetramers. In additional embodiments, the multimers of the invention areat least dimers, at least trimers, or at least tetramers.

[0266] In a preferred embodiment, a TNF-gamma-beta polypeptide of theinvention is a trimer.

[0267] In a highly preferred embodiment, a TNF-gamma-beta polypeptidecomprising, or alternatively consisting of, amino acid residues 72-251of SEQ ID NO:20 and/or amino acid residues 1-181 of SEQ ID NO:26 is atrimer. The subunits of the highly preferred trimer may or may notinclude an amino-terminal methionine residue. In this embodiment, thetrimer consists of, or alternatively comprises, a homomultimer. Also inthis embodiment, the trimer may consist of, or alternatively comprise, aheteromultimer.

[0268] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only TNF-gamma-alpha and/or TNF-gamma-beta polypeptides ofthe invention (including TNF-gamma-alpha and/or TNF-gamma-betafragments, variants, and fusion proteins, as described herein). Thesehomomers may contain TNF-gamma-alpha and/or TNF-gamma-beta polypeptideshaving identical or different amino acid sequences. In a specificembodiment, a homomer of the invention is a multimer containing onlyTNF-gamma-alpha and/or TNF-gamma-beta polypeptides having an identicalamino acid sequence. In another specific embodiment, a homomer of theinvention is a multimer containing TNF-gamma-alpha and/or TNF-gamma-betapolypeptides having different amino acid sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing TNF-gamma-alpha polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing TNF-gamma-alphapolypeptides having identical or different amino acid sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

[0269] As used herein, the term heteromer refers to a multimercontaining heterologous polypeptides (i.e., polypeptides of a differentprotein) in addition to the TNF-gamma-alpha and/or TNF-gamma-betapolypeptides of the invention. In a specific embodiment, the multimer ofthe invention is a heterodimer, a heterotrimer, or a heterotetramer. Inadditional embodiments, the homomeric multimer of the invention is atleast a heterodimer, at least a heterotrimer, or at least aheterotetramer.

[0270] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations. Thus, in oneembodiment, multimers of the invention, such as, for example, homodimersor homotrimers, are formed when polypeptides of the invention contactone another in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentinteractions with and/or between the TNF-gamma-alpha and/orTNF-gamma-beta polypeptides of the invention. Such covalent interactionsmay involve one or more amino acid residues corresponding to thoserecited in SEQ ID NO:2 or SEQ ID NO:20 or SEQ ID NO:26, or correspondingto one or more amino acid residues encoded by the clone HUVEO91, orcorresponding to one or more amino acid residues encoded by the cloneHEMCZ56). Alternatively, such covalent interactions may involve one ormore amino acid residues contained in the heterologous polypeptidesequence in a TNF-gamma-alpha and/or TNF-gamma-beta fusion protein, suchas for example, heterologous sequence contained in a TNF-gamma-alpha-Fcfusion protein (as described herein), and heterologous sequencecontained in a fusion with heterologous polypeptide sequence fromanother TNF family ligand/receptor member, such as, for example,osteoprotegerin, that is capable of forming covalently associatedmultimers.

[0271] The invention also encompasses fusion proteins in which the fulllength TNF-gamma polypeptide or fragment, variant, derivative, or analogthereof is fused to an unrelated protein. Fusion proteins of theinvention may be constructed as direct fusion of TNF-gamma polypeptide(or fragment, variant, derivative, or analog) and a heterologoussequence, or may be constructed with a spacer or adapter region havingone or more amino acids inserted between the two portions of theprotein. Optionally, the spacer region may encode a protease cleavagesite. The precise site of the fusion is not critical and may beroutinely varied by one skilled in the art in order to maximize bindingcharacteristics and/or biological activity of the homologous and/orheterologous sequence(s). The fusion proteins of the invention can beroutinely designed on the basis of the TNF-gamma nucleotide andpolypeptide sequences disclosed herein. For example, as one of skill inthe art will appreciate, TNF-gamma-alpha and/or TNF-gamma-betapolypeptides and fragments (including epitope-bearing fragments) thereofdescribed herein can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric (fusion) polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EP A 394,827; Traunecker, et al, Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric TNF-gamma protein orprotein fragment alone (Fountoulakis, et al., J. Biochem. 270:3958-3964(1995)). As an example, one such TNF-gamma-Fc fusion has been producedherein as described above. In other embodiments, the full lengthTNF-gamma polypeptide or fragment, variant, derivative, or analogthereof is fused to one or more other heterologous polypeptide sequencesthat are capable of forming multimeric formations, such as, for example,the dimerization domain of osteoprotegrin (see, e.g.,. EP 0 721 983,U.S. Pat. No. 5,478,925, and International Publication No. WO 98/49305,each of which is herein incorporated by reference in its entirety).Additional examples of TNF-gamma fusion proteins that are encompassed bythe invention include, but are not limited to, fusion of the TNF-gammapolypeptide sequence to any amino acid sequence that allows the fusionprotein to be displayed on the cell surface; or fusions to an enzyme,fluorescent protein, or luminescent protein which provides a markerfunction.

[0272] Modifications of chimeric OPG polypeptides are encompassed by theinvention and include post-translational modifications (e.g., N-linkedor O-linked carbohydrate chains, processing of N-terminal or C-terminalends), attachment of chemical moieties to the amino acid backbone,chemical modifications of N-linked or O-linked carbohydrate chains, andaddition of an N-terminal methionine residue as a result of prokaryotichost cell expression. The polypeptides may also be modified with adetectable label, such as an enzymatic, fluorescent, isotopic oraffinity label to allow for detection and isolation of the protein.

[0273] Also provided by the invention are chemically modifiedderivatives of OPG which may provide additional advantages such asincreased solubility, stability and circulating time of the polypeptide,or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

[0274] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000,4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0275] As noted above, the polyethylene glycol may have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), thedisclosures of each of which are incorporated herein by reference.

[0276] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

[0277] As suggested above, polyethylene glycol may be attached toproteins via linkage to any of a number of amino acid residues. Forexample, polyethylene glycol can be linked to a protein via covalentbonds to lysine, histidine, aspartic acid, glutamic acid, or cysteineresidues. One or more reaction chemistries may be employed to attachpolyethylene glycol to specific amino acid residues (e.g., lysine,histidine, aspartic acid, glutamic acid, or cysteine) of the protein orto more than one type of amino acid residue (e.g., lysine, histidine,aspartic acid, glutamic acid, cysteine and combinations thereof) of theprotein.

[0278] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0279] As indicated above, pegylation of the proteins of the inventionmay be accomplished by any number of means. For example, polyethyleneglycol may be attached to the protein either directly or by anintervening linker. Linkerless systems for attaching polyethylene glycolto proteins are described in Delgado et al., Crit. Rev. Thera. DrugCarrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol.68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO95/06058; and WO 98/32466, the disclosures of each of which areincorporated herein by reference.

[0280] One system for attaching polyethylene glycol directly to aminoacid residues of proteins without an intervening linker employstresylated MPEG, which is produced by the modification of monmethoxypolyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Uponreaction of protein with tresylated MPEG, polyethylene glycol isdirectly attached to amine groups of the protein. Thus, the inventionincludes protein-polyethylene glycol conjugates produced by reactingproteins of the invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

[0281] Polyethylene glycol can also be attached to proteins using anumber of different intervening linkers. For example, U.S. Pat. No.5,612,460, the entire disclosure of which is incorporated herein byreference, discloses urethane linkers for connecting polyethylene glycolto proteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

[0282] The number of polyethylene glycol moieties attached to eachprotein of the invention (i.e., the degree of substitution) may alsovary. For example, the pegylated proteins of the invention may belinked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, ormore polyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

[0283] The polypeptides of the present invention have uses whichinclude, but are not limited to, molecular weight marker on SDS-PAGEgels or on molecular sieve gel filtration columns using methods wellknown to those of skill in the art.

Functional Activities

[0284] The functional activity of TNF-gamma polypeptides, and fragments,variants derivatives, and analogs thereof, can be assayed by variousmethods.

[0285] For example, in one embodiment where one is assaying for theability to bind or compete with full-length TNF-gamma polypeptide forbinding to anti-TNF-gamma antibody, various immunoassays known in theart can be used, including but not limited to, competitive andnon-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

[0286] In another embodiment, where a TNF-ligand is identified, bindingcan be assayed, e.g., by means well-known in the art. In anotherembodiment, physiological correlates of TNF-gamma binding to itssubstrates (signal transduction) can be assayed.

[0287] In addition, assays described herein (see Examples 5, 6, and9-15, and otherwise known in the art may routinely be applied to measurethe ability of TNF-gamma polypeptides and fragments, variantsderivatives and analogs thereof to elicit TNF-gamma related biologicalactivity (e.g., to inhibit, or alternatively promote, cellproliferation, tumor formation, angiogenesis, NF-kB activation and celladhesion in vitro or in vivo).

[0288] Other methods will be known to the skilled artisan and are withinthe scope of the invention.

Antibodies

[0289] Further polypeptides of the invention relate to antibodies andT-cell antigen receptors (TCR) which immunospecifically bind apolypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Additional polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:20, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Additionalpolypeptides of the invention relate to antibodies and T-cell antigenreceptors (TCR) which immunospecifically bind a polypeptide, polypeptidefragment, or variant of SEQ ID NO:26, and/or an epitope, of the presentinvention (as determined by immunoassays well known in the art forassaying specific antibody-antigen binding). Antibodies of the inventioninclude, but are not limited to, polyclonal, monoclonal, multispecific,human, humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. The term “antibody,” as used herein,refers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds an antigen. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule. In a preferredembodiment, the immunoglobulin is an IgG1 isotype. In a preferredembodiment, the immunoglobulin is an IgG2 isotype. In another preferredembodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins mayhave both a heavy and light chain.

[0290] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Pd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

[0291] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0292] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0293] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. In specific embodiments, antibodies of thepresent invention cross-react with murine, rat and/or rabbit homologs ofhuman proteins and the corresponding epitopes thereof. Antibodies thatdo not bind polypeptides with less than 95%, less than 90%, less than85%, less than 80%, less than 75%, less than 70%, less than 65%, lessthan 60%, less than 55%, and less than 50% identity (as calculated usingmethods known in the art and described herein) to a polypeptide of thepresent invention are also included in the present invention. In aspecific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic polypeptide, orcombination(s) of 2, 3, 4, 5, or more of the specific antigenic and/orimmunogenic polypeptides disclosed herein. Further included in thepresent invention are antibodies which bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, or 10⁻⁵ M. More preferably, antibodies ofthe invention specifically bind TNF-gamma-alpha and/or TNF-gamma-beta orfragments or variants thereof with a dissociation constant or K_(D) lessthan or equal to 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, or 10⁻⁸M.Even more preferably, antibodies of the invention bind specifically bindTNF-gamma-alpha and/or TNF-gamma-beta or fragments or variants thereofwith a dissociation constant or K_(D) less than or equal to 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, ¹⁰⁻¹² M,5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. Theinvention encompasses antibodies that bind TNF-gamma-alpha and/orTNF-gamma-beta with a dissociation constant or K_(D) that is within anyone of the ranges that are between each of the individual recitedvalues.

[0294] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

[0295] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Preferably, antibodies of the presentinvention bind an antigenic epitope disclosed herein, or a portionthereof. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand activity or receptor activity by atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 60%, or at least 50% of the activity in absence ofthe antibody.

[0296] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et a J. Immunol. 160(7):3170-3179 (1998); Prat et al., J.Cell. Sci. 11(Pt2):237-247 (1998; Pitard et al., J. Immunol. Methods205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman etal., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

[0297] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0298] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

[0299] The antibodies of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0300] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to anantigen-of- interest can be produced by various procedures well known inthe art. For example, a polypeptide of the invention can be administeredto various host animals including, but not limited to, rabbits, mice,rats, etc. to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

[0301] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

[0302] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 20 and Example 34).In a non-limiting example, mice can be immunized with a polypeptide ofthe invention or a cell expressing such peptide. Once an immune responseis detected, e.g., antibodies specific for the antigen are detected inthe mouse serum, the mouse spleen is harvested and splenocytes isolated.The splenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

[0303] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0304] Another well known method for producing both polyclonal andmonoclonal human B cell lines is transformation using Epstein Barr Virus(EBV). Protocols for generating EBV-transformed B cell lines arecommonly known in the art, such as, for example, the protocol outlinedin Chapter 7.22 of Current Protocols in Immunology, Coligan et al.,Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in itsentirety by reference herein. The source of B cells for transformationis commonly human peripheral blood, but B cells for transformation mayalso be derived from other sources including, but not limited to, lymphnodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues aregenerally made into single cell suspensions prior to EBV transformation.Additionally, steps may be taken to either physically remove orinactivate T cells (e.g., by treatment with cyclosporin A) in Bcell-containing samples, because T cells from individuals seropositivefor anti-EBV antibodies can suppress B cell immortalization by EBV.

[0305] In general, the sample containing human B cells is innoculatedwith EBV, and cultured for 3-4 weeks. A typical source of EBV is theculture supernatant of the B95-8 cell line (ATCC #VR-1492). Physicalsigns of EBV transformation can generally be seen towards the end of the3-4 week culture period. By phase-contrast microscopy, transformed cellsmay appear large, clear, hairy and tend to aggregate in tight clustersof cells. Initially, EBV lines are generally polyclonal. However, overprolonged periods of cell cultures, EBV lines may become monoclonal orpolyclonal as a result of the selective outgrowth of particular B cellclones. Alternatively, polyclonal EBV transformed lines may be subcloned(e.g., by limiting dilution culture) or fused with a suitable fusionpartner and plated at limiting dilution to obtain monoclonal B celllines. Suitable fusion partners for EBV transformed cell lines includemouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma celllines (human x mouse; e.g., SPAM-8, SBC-H20, and CB-F7), and human celllines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the presentinvention also provides a method of generating polyclonal or monoclonalhuman antibodies against polypeptides of the invention or fragmentsthereof, comprising EBV-transformation of human B cells.

[0306] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0307] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

[0308] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0309] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, whichare incorporated herein by reference in their entirety. Humanizedantibodies are antibody molecules from non-human species antibody thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and a framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

[0310] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0311] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, 6,075,181; and 6,114,598 which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (SanJose, Calif.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

[0312] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0313] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby activate orblock its biological activity.

Polynucleotides Encoding Antibodies

[0314] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2. The invention also encompasses polynucleotides thathybridize under stringent or lower stringency hybridization conditions,e.g., as defined supra, to polynucleotides that encode an antibody,preferably, that specifically binds to a polypeptide of the invention,preferably, an antibody that binds to a polypeptide having the aminoacid sequence of SEQ ID NO:20. The invention also encompassespolynucleotides that hybridize under stringent or lower stringencyhybridization conditions, e.g., as defined supra, to polynucleotidesthat encode an antibody, preferably, that specifically binds to apolypeptide of the invention, preferably, an antibody that binds to apolypeptide having the amino acid sequence of SEQ ID NO:26.

[0315] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0316] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be chemically synthesized orobtained from a suitable source (e.g., an antibody cDNA library, or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody of the invention) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR may then be cloned into replicable cloning vectorsusing any method well known in the art.

[0317] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties ), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0318] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998)for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0319] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0320] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-54 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

[0321] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.Methods of producing antibodies include, but are not limited to,hybridoma technology, EBV transformation, and other methods discussedherein as well as through the use recombinant DNA technology, asdiscussed below.

[0322] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0323] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof, or asingle chain antibody of the invention, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

[0324] A variety of host-expression vector systems may be utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0325] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0326] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0327] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0328] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0329] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0330] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

[0331] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol.3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0332] Vectors which use glutamine synthase (GS) or DIFR as theselectable markers can be amplified in the presence of the drugsmethionine sulphoximine or methotrexate, respectively. An advantage ofglutamine synthase based vectors are the availability of cell lines(e.g., the murine myeloma cell line, NSO) which are glutamine synthasenegative. Glutamine synthase expression systems can also function inglutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO)cells) by providing additional inhibitor to prevent the functioning ofthe endogenous gene. A glutamine synthase expression system andcomponents thereof are detailed in PCT publications: WO87/04462;WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which areincorporated in their entireties by reference herein. Additionally,glutamine synthase expression vectors that may be used according to thepresent invention are commercially available from suppliers, including,for example Lonza Biologics, Inc. (Portsmouth, N.H.). Expression andproduction of monoclonal antibodies using a GS expression system inmurine myeloma cells is described in Bebbington et al., Bio/technology10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995)which are incorporated in their entireties by reference herein.

[0333] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52(1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The codingsequences for the heavy and light chains may comprise cDNA or genomicDNA.

[0334] Once an antibody molecule of the invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

[0335] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

[0336] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0337] As discussed, supra, the polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:2 may be fused or conjugated to the above antibody portions tofacilitate purification. The polypeptides corresponding to apolypeptide, polypeptide fragment, or a variant of SEQ ID NO:20 may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides corresponding to SEQID NO:20 may be fused or conjugated to the above antibody portions tofacilitate purification.

[0338] One reported example describes chimeric proteins consisting ofthe first two domains of the human CD4-polypeptide and various domainsof the constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86(1988). The polypeptides of the present invention fused or conjugated toan antibody having disulfide- linked dimeric structures (due to the IgG)may also be more efficient in binding and neutralizing other molecules,than the monomeric secreted protein or protein fragment alone.(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases,the Fc part in a fusion protein is beneficial in therapy and diagnosis,and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

[0339] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate purification. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

[0340] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium(⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, and ⁹⁷Ru.

[0341] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi or other radioisotopes suchas, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S,⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Y, ¹¹⁷Tin, ¹⁸⁶Re,¹⁸⁸Re and ¹⁶⁶Ho. In specific embodiments, an antibody or fragmentthereof is attached to macrocyclic chelators useful for conjugatingradiometal ions, including but not limited to, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and¹⁵³Sm, to polypeptides. In specific embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA). In other specific embodiments, the DOTA is attached to the anantibody of the invention or fragment thereof via a linker molecule.Examples of linker molecules useful for conjugating DOTA to apolypeptide are commonly known in the art—see, for example, DeNardo etal., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug.Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol.26(8):943-50, 1999 which are hereby incorporated by reference in theirentirety.

[0342] A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II)(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerlydaunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)), andanti-mitotic agents (e.g., vincristine and vinblastine).

[0343] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti- angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0344] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0345] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0346] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0347] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

[0348] The antibodies of the invention may be utilized forimmunophenotyping of cell lines and biological samples. The translationproduct of the gene of the present invention may be useful as a cellspecific marker, or more specifically as a cellular marker that isdifferentially expressed at various stages of differentiation and/ormaturation of particular cell types. Monoclonal antibodies directedagainst a specific epitope, or combination of epitopes, will allow forthe screening of cellular populations expressing the marker. Varioustechniques can be utilized using monoclonal antibodies to screen forcellular populations expressing the marker(s), and include magneticseparation using antibody-coated magnetic beads, “panning” with antibodyattached to a solid matrix (i.e., plate), and flow cytometry (See, e.g.,U.S. Pat. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

[0349] These techniques allow for the screening of particularpopulations of cells, such as might be found with hematologicalmalignancies (i.e. minimal residual disease (MRD) in acute leukemicpatients) and “non-self” cells in transplantations to preventGraft-versus-Host Disease (GVHD). Alternatively, these techniques allowfor the screening of hematopoietic stem and progenitor cells capable ofundergoing proliferation and/or differentiation, as might be found inhuman umbilical cord blood.

Assays for Antibody Binding

[0350] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0351] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trasylol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0352] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., 32P or 125I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0353] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0354] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., 3H or 125I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest conjugated to alabeled compound (e.g., ³H or ¹²⁵I) in the presence of increasingamounts of an unlabeled second antibody.

Therapeutic Uses

[0355] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating one or more of the disclosed diseases, disorders, orconditions. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

[0356] A summary of the ways in which the antibodies of the presentinvention may be used therapeutically includes binding polynucleotidesor polypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

[0357] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

[0358] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents, andanti-retroviral agents). Generally, administration of products of aspecies origin or species reactivity (in the case of antibodies) that isthe same species as that of the patient is preferred. Thus, in apreferred embodiment, human antibodies, fragments derivatives, analogs,or nucleic acids, are administered to a human patient for therapy orprophylaxis.

[0359] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M or 10⁻⁵ M. Morepreferably, antibodies of the invention specifically bindTNF-gamma-alpha and/or TNF-gamma-beta or fragments or variants thereofwith a dissociation constant or KD less than or equal to 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10^(−8 M, or) 10⁻⁸ M. Even more preferably,antibodies of the invention bind specifically bind TNF-gamma-alphaand/or TNF-gamma-beta or fragments or variants thereof with adissociation constant or K_(D) less than or equal to 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. The inventionencompasses antibodies that bind TNF-gamma-alpha and/or TNF-gamma-betawith a dissociation constant or K_(D) that is within any one of theranges that are between each of the individual recited values.

[0360] In particular embodiments, the present invention provides amethod of diagnosing, treating, preventing or ameliorating inflammatorydiseases or disorders comprising, administering to an animal, preferablya human, in which such treatment, prevention or amelioration is desiredan antibody that specifically binds TNF-gamma-alpha and/orTNF-gamma-beta (or a TNF-gamma-alpha and/or TNF-gamma-beta antagonistsuch as a DR3- or TR6-Fc fusion protein, see Example 35) or fragment orvariant thereof in an amount effective to treat, prevent or amelioratethe inflammatory disease or disorder. In additional specificembodiments, the inflammatory disease or disorder is inflammatory boweldisease. In additional specific embodiments, the inflammatory disease ordisorder is encephalitis. In additional specific embodiments, theinflammatory disease or disorder is atherosclerosis. In specificembodiments, the inflammatory disease or disorder is psoriasis. Thepresent invention further provides compositions comprising theanti-TNF-gamma-alpha and/or anti-TNF-gamma-beta antibodies and a carrierfor use in the above-described method of diagnosing, treating,preventing or ameliorating inflammatory diseases and disorders.

[0361] In specific embodiments, the present invention provides a methodof diagnosing, treating, preventing or ameliorating inflammationcomprising administering to an animal, preferably a human, in which suchtreatment, prevention or amelioration is desired an antibody thatspecifically binds TNF-gamma-alpha and/or TNF-gamma-beta (or aTNF-gamma-alpha and/or TNF-gamma-beta antagonist such as a DR3- orTR6-Fc fusion protein, see Example 35) or fragment or variant thereof inan amount effective to treat, prevent or ameliorate the inflammation.The present invention further provides compositions comprising theanti-TNF-gamma-alpha and/or anti-TNF-gamma-beta antibodies and a carrierfor use in the above-described method of diagnosing, treating,preventing or ameliorating inflammation.

[0362] In specific embodiments, the present invention provides a methodof diagnosing, treating, preventing or ameliorating graft versus hostdisease (GVHD) comprising administering to an animal, preferably ahuman, in which such treatment, prevention or amelioration is desired anantibody that specifically binds TNF-gamma-alpha and/or TNF-gamma-beta(or a TNF-gamma-alpha and/or TNF-gamma-beta antagonist such as a DR3- orTR6-Fc fusion protein, see Example 35) or fragment or variant thereof inan amount effective to treat, prevent or ameliorate the GVHD. Thepresent invention further provides compositions comprising theanti-TNF-gamma-alpha and/or anti-TNF-gamma-beta antibodies and a carrierfor use in the above-described method of diagnosing, treating,preventing or ameliorating GVHD.

[0363] In other embodiments, the present invention provides a method ofdiagnosing, treating, preventing or ameliorating autoimmune diseases anddisorders comprising administering to an animal, preferably a human, inwhich such treatment, prevention or amelioration is desired an antibodythat specifically binds TNF-gamma-alpha and/or TNF-gamma-beta (or aTNF-gamma-alpha and/or TNF-gamma-beta antagonist such as a DR3- orTR6-Fc fusion protein, see Example 35) or fragment or variant thereof inan amount effective to treat, prevent or ameliorate the autoimmunedisease or disorder. In specific embodiments, the autoimmune disease ordisorder is systemic lupus erythematosus. In specific embodiments, theautoimmune disease or disorder is arthritis, particularly rheumatoidarthritis. In specific embodiments, the autoimmune disease or disorderis multiple sclerosis. In specific embodiments, the autoimmune diseaseor disorder is Crohn's disease. In specific embodiments, the autoimmunedisease or disorder is autoimmune encephalitis. The present inventionfurther provides compositions comprising the anti-TNF-gamma-alpha and/oranti-TNF-gamma-beta antibodies and a carrier for use in theabove-described method of diagnosing, treating, preventing orameliorating autoimmune diseases and disorders.

[0364] In specific embodiments, the present invention provides a methodof diagnosing, treating, preventing or ameliorating allergy or asthmacomprising administering to an animal, preferably a human, in which suchtreatment, prevention or amelioration is desired an antibody thatspecifically binds TNF-gamma-alpha and/or TNF-gamma-beta (or aTNF-gamma-alpha and/or TNF-gamma-beta antagonist such as a DR3- orTR6-Fc fusion protein, see Example 35) or fragment or variant thereof inan amount effective to treat, prevent or ameliorate the allergy orasthma. The present invention further provides compositions comprisingthe anti-TNF-gamma-alpha and/or anti-TNF-gamma-beta antibodies and acarrier for use in the above-described method of diagnosing, treating,preventing or ameliorating allergy or asthma.

[0365] The present invention further encompasses methods andcompositions for reducing Tcell activation, comprising contacting aneffective amount of anti-TNF-gamma-alpha and/or anti-TNF-gamma-betaantibody (or other TNF-gamma-alpha and/or TNF-gamma-beta antagonist suchas a DR3- or TR6-Fc fusion protein see Example 35) with cells ofhematopoietic origin, wherein the effective amount of theanti-TNF-gamma-alpha and/or anti-TNF-gamma-beta antibody reduces T cellactivation. In preferred embodiments, the cells of hematopoietic originare T cells. In other preferred embodiments, the effective amount of theanti-TNF-gamma-alpha and/or anti-TNF-gamma-beta antibody reducesTNF-gamma-alpha and/or TNF-gamma beta induced T cell activation.

[0366] The present invention further encompasses methods andcompositions for reducing Tcell activation comprising, or alternativelyconsisting of, administering to an animal, preferably a human, in whichsuch reduction is desired, an antibody that specifically bindsTNF-gamma-alpha and/or TNF-gamma-beta (or a TNF-gamma-alpha and/orTNF-gamma-beta antagonist such as a DR3- or TR6-Fc fusion protein, seeExample 35) or fragment or variant thereof in an amount effective toreduce T cell activation. The present invention further providescompositions comprising the anti-TNF-gamma-alpha and/oranti-TNF-gamma-beta antibodies and a carrier for use in theabove-described method of reducing T cell activation.

Gene Therapy

[0367] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0368] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0369] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

[0370] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

[0371] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0372] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;W092/20316; W093/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

[0373] In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

[0374] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationW094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0375] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0376] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0377] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0378] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0379] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0380] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0381] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

[0382] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

[0383] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

[0384] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention, preferably anantibody of the invention. In a preferred aspect, the compound issubstantially purified (e.g., substantially free from substances thatlimit its effect or produce undesired side-effects). The subject ispreferably an animal, including but not limited to animals such as cows,pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal,and most preferably human.

[0385] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0386] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0387] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0388] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0389] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev.Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,i.e., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

[0390] Other controlled release systems are discussed in the review byLanger (Science 249:1527-1533 (1990)).

[0391] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0392] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0393] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0394] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0395] The amount of the compound of the invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0396] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0397] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

Diagnosis and Imaging

[0398] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

[0399] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0400] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium ³H), indium (¹¹²In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0401] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0402] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells which contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

[0403] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0404] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0405] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0406] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

[0407] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0408] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope whichis specifically immunoreactive with at least one anti-polypeptideantigen antibody. Further, such a kit includes means for detecting thebinding of said antibody to the antigen (e.g., the antibody may beconjugated to a fluorescent compound such as fluorescein or rhodaminewhich can be detected by flow cytometry). In specific embodiments, thekit may include a recombinantly produced or chemically synthesizedpolypeptide antigen. The polypeptide antigen of the kit may also beattached to a solid support.

[0409] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0410] In an additional embodiment, the invention includes a diagnostickit for use in screening serum containing antigens of the polypeptide ofthe invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0411] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0412] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0413] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Transgenics

[0414] The polypeptides of the invention can also be expressed intransgenic animals. Animals of any species, including, but not limitedto, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats,sheep, cows and non-human primates, e.g., baboons, monkeys, andchimpanzees may be used to generate transgenic animals. In a specificembodiment, techniques described herein or otherwise known in the art,are used to express polypeptides of the invention in humans, as part ofa gene therapy protocol.

[0415] Any technique known in the art may be used to introduce thetransgene (i.e., polynucleotides of the invention) into animals toproduce the founder lines of transgenic animals. Such techniquesinclude, but are not limited to, pronuclear microinjection ((each of thefollowing references is hereby incorporated by reference) Paterson etal., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al.,Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., Biotechnology(NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191(1989)); retrovirus mediated gene transfer into germ lines ((thefollowing reference is hereby incorporated by reference) Van der Puttenet al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts orembryos; gene targeting in embryonic stem cells ((each of the followingreferences is hereby incorporated by reference) Thompson et al., Cell56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, MolCell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides ofthe invention using a gene gun ((the following reference is herebyincorporated by reference) see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pluripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer ((the following reference is herebyincorporated by reference) Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

[0416] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence ((each of the followingreferences is hereby incorporated by reference) Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

[0417] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. ((the following reference is herebyincorporated by reference) Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. ((the following reference is herebyincorporated by reference) Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

[0418] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[0419] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[0420] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of TNF-gamma-alpha and/orTNF-gamma-beta polypeptides, studying conditions and/or disordersassociated with aberrant TNF-gamma-alpha and/or TNF-gamma-betaexpression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

[0421] Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. ((each of the following references is hereby incorporatedby reference) E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene ((each of the followingreferences is hereby incorporated by reference) e.g., see Thomas &Capecchi 1987 and Thompson 1989, supra). However this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art.

[0422] In further embodiments of the invention, cells that aregenetically engineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

[0423] Alternatively, the cells can be incorporated into a matrix andimplanted in the body, e.g., genetically engineered fibroblasts can beimplanted as part of a skin graft; genetically engineered endothelialcells can be implanted as part of a lymphatic or vascular graft. ((eachof the following references is hereby incorporated by reference) See,for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan &Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated byreference herein in its entirety).

[0424] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[0425] In a specific embodiment, a transgenic expression construct wasgenerated using the pAC vector to express amino acid residues T-28through L-174 of SEQ ID NO:2. In another specific embodiment, atransgenic expression construct was generated using the pTR vector toexpress amino acid residues T-28 through L-174 of SEQ ID NO:2.

Diagnostics

[0426] The present inventors have discovered that TNF-gamma is expressedin human umbilical vein endothelial cells, induced endothelial cells,macrophages, and substantia nigra tissue. For a number of immune andcirculatory systems-related disorders, substantially altered (increasedor decreased) levels of TNF-gamma-alpha and/or TNF-gamma-beta geneexpression can be detected in immune and circulatory systems tissue orother cells or bodily fluids (e.g., sera, plasma, urine, synovial fluidor spinal fluid) taken from an individual having such a disorder,relative to a “standard” TNF-gamma-alpha and/or TNF-gamma-beta geneexpression level, that is, the TNF-gamma-alpha and/or TNF-gamma-betaexpression level in immune and circulatory systems tissues or bodilyfluids from an individual not having the immune and circulatory systemsdisorder. Thus, the invention provides a diagnostic method useful duringdiagnosis of a immune and circulatory systems disorder, which involvesmeasuring the expression level of the gene encoding the TNF-gamma-alphaand/or TNF-gamma-beta protein in immune and circulatory systems tissueor other cells or body fluid from an individual and comparing themeasured gene expression level with a standard TNF-gamma-alpha and/orTNF-gamma-beta gene expression level, whereby an increase or decrease inthe gene expression level compared to the standard is indicative of animmune and circulatory systems disorder.

[0427] In particular, it is believed that certain tissues in mammalswith cancer of the immune and circulatory systems express significantlyreduced levels of the TNF-gamma-alpha and/or TNF-gamma-beta protein andmRNA encoding the TNF-gamma-alpha and/or TNF-gamma-beta protein whencompared to a corresponding “standard” level. Further, it is believedthat enhanced levels of the TNF-gamma-alpha and/or TNF-gamma-betaprotein can be detected in certain body fluids (e.g., sera, plasma,urine, and spinal fluid) from mammals with such a cancer when comparedto sera from mammals of the same species not having the cancer.

[0428] Thus, the invention provides a diagnostic method useful duringdiagnosis of a immune and circulatory systems disorder, includingcancers of these systems, which involves measuring the expression levelof the gene encoding the TNF-gamma-alpha and/or TNF-gamma-beta proteinin immune and circulatory systems tissue or other cells or body fluidfrom an individual and comparing the measured gene expression level witha standard TNF-gamma-alpha and/or TNF-gamma-beta gene expression level,whereby an increase or decrease in the gene expression level compared tothe standard is indicative of an immune and circulatory systemsdisorder.

[0429] Where a diagnosis of a disorder in the immune and circulatorysystems, including diagnosis of a tumor, has already been made accordingto conventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting depressed TNF-gamma-alpha and/orTNF-gamma-beta gene expression will experience a worse clinical outcomerelative to patients expressing the gene at a level nearer the standardlevel.

[0430] By “assaying the expression level of the gene encoding theTNF-gamma-alpha and/or TNF-gamma-beta protein” is intended qualitativelyor quantitatively measuring or estimating the level of theTNF-gamma-alpha and/or TNF-gamma-beta protein or the level of the mRNAencoding the TNF-gamma-alpha and/or TNF-gamma-beta protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level or mRNA level) or relatively (e.g., by comparingto the TNF-gamma-alpha and/or TNF-gamma-beta protein level or mRNA levelin a second biological sample). Preferably, the TNF-gamma-alpha and/orTNF-gamma-beta protein level or mRNA level in the first biologicalsample is measured or estimated and compared to a standardTNF-gamma-alpha and/or TNF-gamma-beta protein level or mRNA level, thestandard being taken from a second biological sample obtained from anindividual not having the disorder or being determined by averaginglevels from a population of individuals not having a disorder of theimmune and circulatory systems. As will be appreciated in the art, oncea standard TNF-gamma-alpha and/or TNF-gamma-beta protein level or mRNAlevel is known, it can be used repeatedly as a standard for comparison.

[0431] By “biological sample” is intended any biological sample obtainedfrom an individual, body fluid, cell line, tissue culture, or othersource which contains TNF-gamma-alpha and/or TNF-gamma-beta protein ormRNA. As indicated, biological samples include body fluids (such assera, plasma, urine, synovial fluid and spinal fluid) which contain freeTNF-gamma-alpha and/or TNF-gamma-beta protein, immune and circulatorysystems tissue, and other tissue sources found to express complete ormature TNF-gamma-alpha and/or TNF-gamma-beta or a TNF-gamma-alpha and/orTNF-gamma-beta receptor. Methods for obtaining tissue biopsies and bodyfluids from mammals are well known in the art. Where the biologicalsample is to include mRNA, a tissue biopsy is the preferred source.

[0432] Total cellular RNA can be isolated from a biological sample usingany suitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described byChomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)). Levels ofmRNA encoding the TNF-gamma-alpha and/or TNF-gamma-beta protein are thenassayed using any appropriate method. These include Northern blotanalysis, S1 nuclease mapping, the polymerase chain reaction (PCR),reverse transcription in combination with the polymerase chain reaction(RT-PCR), and reverse transcription in combination with the ligase chainreaction (RT-LCR).

[0433] Assaying TNF-gamma-alpha and/or TNF-gamma-beta protein levels ina biological sample can occur using antibody-based techniques. Forexample, TNF-gamma-alpha and/or TNF-gamma-beta protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting TNF-gamma-alpha and/or TNF-gamma-betaprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium (^(99m)Tc), and fluorescent labels, such as fluorescein andrhodamine, and biotin.

[0434] In addition to assaying TNF-gamma-alpha and/or TNF-gamma-betaprotein levels in a biological sample obtained from an individual,TNF-gamma-alpha and/or TNF-gamma-beta protein can also be detected invivo by imaging. Antibody labels or markers for in vivo imaging ofTNF-gamma-alpha and/or TNF-gamma-beta protein include those detectableby X-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

[0435] A TNF-gamma-alpha and/or TNF-gamma-beta protein-specific antibodyor antibody fragment which has been labeled with an appropriatedetectable imaging moiety, such as a radioisotope (for example, ¹³¹I,¹¹²In, ^(99m)Tc), a radio-opaque substance, or a material detectable bynuclear magnetic resonance, is introduced (for example, parenterally,subcutaneously or intraperitoneally) into the mammal to be examined forimmune system disorder. It will be understood in the art that the sizeof the subject and the imaging system used will determine the quantityof imaging moiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain TNF-gamma-alpha and/orTNF-gamma-beta protein. In vivo tumor imaging is described by Burchieland coworkers (Chapter 13 in Tumor Imaging: The Radiochemical Detectionof Cancer, Burchiel, S. W. and Rhodes, B. A., eds., Masson PublishingInc. (1982)).

Therapeutics

[0436] As noted above, TNF-gamma-alpha and/or TNF-gamma-betapolynucleotides and polypeptides are useful for diagnosis of conditionsinvolving abnormally high or low expression of TNF-gamma-alpha and/orTNF-gamma-beta activities. Given the cells and tissues whereTNF-gamma-alpha and/or TNF-gamma-beta is expressed as well as theactivities modulated by TNF-gamma-alpha and/or TNF-gamma-beta, it isreadily apparent that a substantially altered (increased or decreased)level of expression of TNF-gamma-alpha and/or TNF-gamma-beta in anindividual compared to the standard or “normal” level producespathological conditions related to the bodily system(s) in whichTNF-gamma-alpha and/or TNF-gamma-beta is expressed and/or is active.

[0437] It will also be appreciated by one of ordinary skill that, sincethe TNF-gamma-alpha and/or TNF-gamma-beta proteins of the invention aremembers of the TNF family the mature secreted form of the protein may bereleased in soluble form from the cells which express TNF-gamma byproteolytic cleavage. Therefore, when TNF-gamma-alpha and/orTNF-gamma-beta mature form or soluble extracellular domain is added froman exogenous source to cells, tissues or the body of an individual, theprotein will exert its physiological activities on its target cells ofthat individual. Also, cells expressing this type II transmembraneprotein may be added to cells, tissues or the body of an individual andthese added cells will bind to cells expressing receptor forTNF-gamma-alpha and/or TNF-gamma-beta, whereby the cells expressingTNF-gamma-alpha and/or TNF-gamma-beta can cause actions (e.g. regulationof endothelial cell growth and regulation) on the receptor-bearingtarget cells.

[0438] Therefore, it will be appreciated that conditions caused by adecrease in the standard or normal level of TNF-gamma-alpha and/orTNF-gamma-beta activities in an individual, particularly disorders ofthe immune and circulatory systems, can be treated, prevented,diagnosed, and/or detected by administration of TNF-gamma-alpha and/orTNF-gamma-beta polypeptide (in the form of the mature protein). Thus,the invention also provides a method of treatment, prevention,diagnosis, and/or detection of an individual in need of an increasedlevel of TNF-gamma-alpha and/or TNF-gamma-beta activity comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated TNF-gamma-alpha and/orTNF-gamma-beta polypeptide of the invention, particularly a mature formof the TNF-gamma-alpha and/or TNF-gamma-beta protein of the invention,effective to increase the TNF-gamma-alpha and/or TNF-gamma-beta activitylevel in such an individual.

[0439] Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof are useful in the treatment,prevention, diagnosis, and/or detection of a wide range of diseasesand/or conditions. Such diseases and conditions include, but are notlimited to, cancer (e.g., immune cell related cancers, breast cancer,prostate cancer, ovarian cancer, follicular lymphoma, cancer associatedwith mutation or alteration of p53, brain tumor, bladder cancer,uterocervical cancer, colon cancer, colorectal cancer, non-small cellcarcinoma of the lung, small cell carcinoma of the lung, stomach cancer,etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial(e.g., viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2infection, herpesvirus infection (including, but not limited to, HSV-1,HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirusinfection, human papilloma virus infection, hepatitis infection (e.g.,HAV, HBV, HCV, etc.), Helicobacter pylori infection, invasiveStaphylococcia, etc.), parasitic infection, nephritis, bone disease(e.g., osteoporosis), atherosclerosis, pain, cardiovascular disorders(e.g., neovascularization, hypovascularization or reduced circulation(e.g., ischemic disease (e.g., myocardial infarction, stroke, etc.)),AIDS, allergy, inflammation, neurodegenerative disease (e.g.,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,pigmentary retinitis, cerebellar degeneration, etc.), dementia, graftrejection (acute and chronic), graft vs. host disease, diseases due toosteomyelodysplasia (e.g., aplastic anemia, etc.), joint tissuedestruction in rheumatism, liver disease (e.g., acute and chronichepatitis, liver injury, and cirrhosis), autoimmune disease (e.g.,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,immune complex glomerulonephritis, autoimmune diabetes, autoimmunethrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis,etc.), cardiomyopathy (e.g., dilated cardiomyopathy), diabetes, diabeticcomplications (e.g., diabetic nephropathy, diabetic neuropathy, diabeticretinopathy), influenza, asthma, psoriasis, glomerulonephritis, septicshock, and ulcerative colitis.

[0440] Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, diagnosing and/or prognosing diseases, disorders, and/orconditions of/associated with the immune system, by, for example,activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immune systemassociated diseases, disorders, and/or conditions may be genetic,somatic, such as cancer and some autoimmune diseases, acquired (e.g., bychemotherapy or toxins), or infectious. Moreover, polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention can be used as a marker or detector of a particular immunesystem disease or disorder.

[0441] In another embodiment, a polypeptide of the invention, orpolynucleotides, antibodies, agonists, or antagonists corresponding tothat polypeptide, may be used to treat diseases and disorders of theimmune system and/or to inhibit or enhance an immune response generatedby cells associated with the tissue(s) in which the polypeptide of theinvention is expressed.

[0442] Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, diagnosing, and/or prognosing immunodeficiencies, includingboth congenital and acquired immunodeficiencies. Examples of B cellimmunodeficiencies in which immunoglobulin levels B cell function and/orB cell numbers are decreased include: X-linked agammaglobulinemia(Bruton's disease), X-linked infantile agammaglobulinemia, X-linkedimmunodeficiency with hyper IgM, non X-linked immunodeficiency withhyper IgM, X-linked lymphoproliferative syndrome (XLP),agammaglobulinemnia including congenital and acquiredagammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swisstype), Selective IgM deficiency, selective IgA deficiency, selective IgGsubclass deficiencies, IgG subclass deficiency (with or without IgAdeficiency), Ig deficiency with increased IgM, IgG and IgA deficiencywith increased IgM, antibody deficiency with normal or elevated Igs, Igheavy chain deletions, kappa chain deficiency, B celllymphoproliferative disorder (BLPD), common variable immunodeficiency(CVID), common variable immunodeficiency (CVI) (acquired), and transienthypogammaglobulinemia of infancy.

[0443] In specific embodiments, ataxia-telangiectasia or conditionsassociated with ataxia-telangiectasia are treated, prevented, diagnosed,and/or prognosing using the polypeptides or polynucleotides of theinvention, and/or agonists or antagonists thereof.

[0444] Examples of congenital immunodeficiencies in which T cell and/orB cell function and/or number is decreased include, but are not limitedto: DiGeorge anomaly, severe combined immunodeficiencies (SCID)(including, but not limited to, X-linked SCID, autosomal recessive SCID,adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP)deficiency, Class II MHC deficiency (Bare lymphocyte syndrome),Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia,third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronicmucocutaneous candidiasis, natural killer cell deficiency (NK),idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant Tcell defect (unspecified), and unspecified immunodeficiency of cellmediated immunity.

[0445] In specific embodiments, DiGeorge anomaly or conditionsassociated with DiGeorge anomaly are treated, prevented, diagnosed,and/or prognosed using polypeptides or polynucleotides of the invention,or antagonists or agonists thereof.

[0446] Other immunodeficiencies that may be treated, prevented,diagnosed, and/or prognosed using polypeptides or polynucleotides of theinvention, and/or agonists or antagonists thereof, include, but are notlimited to, chronic granulomatous disease, Chediak-Higashi syndrome,myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenasedeficiency, X-linked lymphoproliferative syndrome (XLP), leukocyteadhesion deficiency, complement component deficiencies (including C1,C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticulardysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma,severe congenital leukopenia, dysplasia with immunodeficiency, neonatalneutropenia, short limbed dwarfism, and Nezelof syndrome-combinedimmunodeficiency with Igs.

[0447] In a preferred embodiment, the immunodeficiencies and/orconditions associated with the immunodeficiencies recited above aretreated, prevented, diagnosed and/or prognosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention.

[0448] In a preferred embodiment polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used as an agent to boost immunoresponsiveness amongimmunodeficient individuals. In specific embodiments, polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention could be used as an agent to boost immunoresponsiveness amongB cell and/or T cell immunodeficient individuals.

[0449] The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, diagnosing and/or prognosing autoimmune disorders. Manyautoimmune disorders result from inappropriate recognition of self asforeign material by immune cells. This inappropriate recognition resultsin an immune response leading to the destruction of the host tissue.Therefore, the administration of polynucleotides and polypeptides of theinvention that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

[0450] Autoimmune diseases or disorders that may be treated, prevented,diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention include, but arenot limited to, one or more of the following: systemic lupuserythematosus, rheumatoid arthritis, ankylosing spondylitis, multiplesclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmunehemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmunethrombocytopenia purpura, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenleinpurpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigusvulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), andinsulin-resistant diabetes mellitus.

[0451] Additional disorders that are likely to have an autoimmunecomponent that may be treated, prevented, and/or diagnosed with thecompositions of the invention include, but are not limited to, type IIcollagen-induced arthritis, antiphospholipid syndrome, dermatitis,allergic encephalomyelitis, myocarditis, relapsing polychondritis,rheumatic heart disease, neuritis, uveitis ophthalmia,polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmunepulmonary inflammation, autism, Guillain-Barre Syndrome, insulindependent diabetes mellitus, and autoimmune inflammatory eye disorders.

[0452] Additional disorders that are likely to have an autoimmunecomponent that may be treated, prevented, diagnosed and/or prognosedwith the compositions of the invention include, but are not limited to,scleroderma with anti-collagen antibodies (often characterized, e.g., bynucleolar and other nuclear antibodies), mixed connective tissue disease(often characterized, e.g., by antibodies to extractable nuclearantigens (e.g., ribonucleoprotein)), polymyositis (often characterized,e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g.,by antiparietal cell, microsomes, and intrinsic factor antibodies),idiopathic Addison's disease (often characterized, e.g., by humoral andcell-mediated adrenal cytotoxicity, infertility (often characterized,e.g., by antispermatozoal antibodies), glomerulonephritis (oftencharacterized, e.g., by glomerular basement membrane antibodies orimmune complexes), bullous pemphigoid (often characterized, e.g., by IgGand complement in basement membrane), Sjogren's syndrome (oftencharacterized, e.g., by multiple tissue antibodies, and/or a specificnonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., bycell-mediated and humoral islet cell antibodies), and adrenergic drugresistance (including adrenergic drug resistance with asthma or cysticfibrosis) (often characterized, e.g., by beta-adrenergic receptorantibodies).

[0453] Additional disorders that may have an autoimmune component thatmay be treated, prevented, diagnosed and/or prognosed with thecompositions of the invention include, but are not limited to, chronicactive hepatitis (often characterized, e.g., by smooth muscleantibodies), primary biliary cirrhosis (often characterized, e.g., bymitochondria antibodies), other endocrine gland failure (oftencharacterized, e.g., by specific tissue antibodies in some cases),vitiligo (often characterized, e.g., by melanocyte antibodies),vasculitis (often characterized, e.g., by Ig and complement in vesselwalls and/or low serum complement), post-MI (often characterized, e.g.,by myocardial antibodies), cardiotomy syndrome (often characterized,e.g., by myocardial antibodies), urticaria (often characterized, e.g.,by IgG and IgM antibodies to IgE), atopic dermatitis (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), asthma (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), and many otherinflammatory, granulomatous, degenerative, and atrophic disorders.

[0454] In a preferred embodiment, the autoimmune diseases and disordersand/or conditions associated with the diseases and disorders recitedabove are treated, prevented, diagnosed and/or prognosed using forexample, antagonists or agonists, polypeptides or polynucleotides, orantibodies of the present invention. In a specific preferred embodiment,rheumatoid arthritis is treated, prevented, and/or diagnosed usingpolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention.

[0455] In another specific preferred embodiment, systemic lupuserythematosus is treated, prevented, and/or diagnosed usingpolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention. In another specific preferredembodiment, idiopathic thrombocytopenia purpura is treated, prevented,and/or diagnosed using polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention.

[0456] In another specific preferred embodiment IgA nephropathy istreated, prevented, and/or diagnosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention.

[0457] In a preferred embodiment, the autoimmune diseases and disordersand/or conditions associated with the diseases and disorders recitedabove are treated, prevented, diagnosed and/or prognosed usingpolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention

[0458] In preferred embodiments, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an immunosuppressive agent(s).

[0459] Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, prognosing, and/or diagnosing diseases, disorders, and/orconditions of hematopoietic cells. Polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with a decrease in certain (or many) types hematopoieticcells, including but not limited to, leukopenia, neutropenia, anemia,and thrombocytopenia. Alternatively, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventioncould be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat or prevent those diseases, disorders, and/or conditionsassociated with an increase in certain (or many) types of hematopoieticcells, including but not limited to, histiocytosis.

[0460] Allergic reactions and conditions, such as asthma (particularlyallergic asthma) or other respiratory problems, may also be treated,prevented, diagnosed and/or prognosed using polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof. Moreover, these molecules can be used to treat, prevent,prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenicmolecule, or blood group incompatibility.

[0461] Additionally, polypeptides or polynucleotides of the invention,and/or agonists or antagonists thereof, may be used to treat, prevent,diagnose and/or prognose IgE-mediated allergic reactions. Such allergicreactions include, but are not limited to, asthma, rhinitis, and eczema.In specific embodiments, polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be used tomodulate IgE concentrations in vitro or in vivo.

[0462] Moreover, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention have uses in thediagnosis, prognosis, prevention, and/or treatment of inflammatoryconditions. For example, since polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagonists of theinvention may inhibit the activation, proliferation and/ordifferentiation of cells involved in an inflammatory response, thesemolecules can be used to prevent and/or treat chronic and acuteinflammatory conditions. Such inflammatory conditions include, but arenot limited to, for example, inflammation associated with infection(e.g., septic shock, sepsis, or systemic inflammatory responsesyndrome), ischemia-reperfusion injury, endotoxin lethality,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, over production of cytokines (e.g., TNF or IL-1.), respiratorydisorders (e.g., asthma and allergy); gastrointestinal disorders (e.g.,inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung,bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis;ischemic brain injury and/or stroke, traumatic brain injury,neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer'sdisease); AIDS-related dementia; and prion disease); cardiovasculardisorders (e.g., atherosclerosis, myocarditis, cardiovascular disease,and cardiopulmonary bypass complications); as well as many additionaldiseases, conditions, and disorders that are characterized byinflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma,pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusioninjury, Grave's disease, systemic lupus erythematosus, diabetesmellitus, and allogenic transplant rejection).

[0463] Because inflammation is a fundamental defense mechanism,inflammatory disorders can effect virtually any tissue of the body.Accordingly, polynucleotides, polypeptides, and antibodies of theinvention, as well as agonists or antagonists thereof, have uses in thetreatment of tissue-specific inflammatory disorders, including, but notlimited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis,balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis,cervicitis, cholecystitis, chorditis, cochlitis, colitis,conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis,endocarditis, esophagitis, eustachitis, fibrositis, folliculitis,gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis,keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, mediaotitis, meningitis, metritis, mucitis, myocarditis, myosititis,myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis,pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis,poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis,scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis,steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis,urethritis, and vaginitis.

[0464] In specific embodiments, polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, are useful to diagnose, prognose, prevent, and/or treat organtransplant rejections and graft-versus-host disease. Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. Polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, that inhibit an immune response, particularly the activation,proliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing organ rejection or GVHD. In specificembodiments, polypeptides, antibodies, or polynucleotides of theinvention, and/or agonists or antagonists thereof, that inhibit animmune response, particularly the activation, proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing experimental allergic and hyperacute xenograft rejection.

[0465] In other embodiments, polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagoniststhereof, are useful to diagnose, prognose, prevent, and/or treat immunecomplex diseases, including, but not limited to, serum sickness, poststreptococcal glomerulonephritis, polyarteritis nodosa, and immunecomplex-induced vasculitis.

[0466] Polypeptides, antibodies, polynucleotides and/or agonists orantagonists of the invention can be used to treat, detect, and/orprevent infectious agents. For example, by increasing the immuneresponse, particularly increasing the proliferation activation and/ordifferentiation of B and/or T cells, infectious diseases may be treated,detected, and/or prevented. The immune response may be increased byeither enhancing an existing immune response, or by initiating a newimmune response. Alternatively, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention mayalso directly inhibit the infectious agent (refer to section ofapplication listing infectious agents, etc), without necessarilyeliciting an immune response.

[0467] In another embodiment, polypeptides, antibodies, polynucleotidesand/or agonists or antagonists of the present invention are used as avaccine adjuvant that enhances immune responsiveness to an antigen. In aspecific embodiment, polypeptides, antibodies, polynucleotides and/oragonists or antagonists of the present invention are used as an adjuvantto enhance tumor-specific immune responses.

[0468] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an adjuvant to enhance anti-viral immune responses.Anti-viral immune responses that may be enhanced using the compositionsof the invention as an adjuvant, include virus and virus associateddiseases or symptoms described herein or otherwise known in the art. Inspecific embodiments, the compositions of the invention are used as anadjuvant to enhance an immune response to a virus, disease, or symptomselected from the group consisting of: AIDS, meningitis, Dengue, EBV,and hepatitis (e.g., hepatitis B). In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus,Japanese B encephalitis, influenza A and B, parainfluenza, measles,cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpessimplex, and yellow fever.

[0469] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an adjuvant to enhance anti-bacterial or anti-fungal immuneresponses. Anti-bacterial or anti-fungal immune responses that may beenhanced using the compositions of the invention as an adjuvant, includebacteria or fungus and bacteria or fungus associated diseases orsymptoms described herein or otherwise known in the art. In specificembodiments, the compositions of the invention are used as an adjuvantto enhance an immune response to a bacteria or fungus, disease, orsymptom selected from the group consisting of: tetanus, Diphtheria,botulism, and meningitis type B.

[0470] In another specific embodiment, the compositions of the inventionare used as an adjuvant to enhance an immune response to a bacteria orfungus, disease, or symptom selected from the group consisting of:Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonellaparatyphi, Neisseria meningitidis, Streptococcus pneumoniae, Group Bstreptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, and Borrelia burgdorferi.

[0471] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an adjuvant to enhance anti-parasitic immune responses.Anti-parasitic immune responses that may be enhanced using thecompositions of the invention as an adjuvant, include parasite andparasite associated diseases or symptoms described herein or otherwiseknown in the art. In specific embodiments, the compositions of theinvention are used as an adjuvant to enhance an immune response to aparasite. In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response toPlasmodium (malaria) or Leishmania.

[0472] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay also be employed to treat infectious diseases including silicosis,sarcoidosis, and idiopathic pulmonary fibrosis; for example, bypreventing the recruitment and activation of mononuclear phagocytes.

[0473] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an antigen for the generation of antibodies to inhibit orenhance immune mediated responses mediated by polypeptides of theinvention.

[0474] In one embodiment, polypeptides, antibodies, polynucleotidesand/or agonists or antagonists of the present invention are administeredto an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs,micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-humanprimate, and human, most preferably human) to boost the immune system toproduce increased quantities of one or more antibodies (e.g., IgG, IgA,IgM, and IgE), to induce higher affinity antibody production andimmunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or toincrease an immune response.

[0475] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a stimulator of B cell responsiveness to pathogens.

[0476] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an activator of T cells.

[0477] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an inhibitor of T cell function.

[0478] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent that elevates the immune status of an individualprior to their receipt of immunosuppressive therapies.

[0479] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to induce higher affinity antibodies.

[0480] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to increase serum immunoglobulin concentrations.

[0481] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to accelerate recovery of immunocompromisedindividuals.

[0482] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to boost immunoresponsiveness among agedpopulations and/or neonates.

[0483] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an immune system enhancer prior to, during, or after bonemarrow transplant and/or other transplants (e.g., allogeneic orxenogeneic organ transplantation). With respect to transplantation,compositions of the invention may be administered prior to, concomitantwith, and/or after transplantation. In a specific embodiment,compositions of the invention are administered after transplantation,prior to the beginning of recovery of T-cell populations. In anotherspecific embodiment, compositions of the invention are firstadministered after transplantation after the beginning of recovery of Tcell populations, but prior to full recovery of B cell populations.

[0484] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to boost immunoresponsiveness among individualshaving an acquired loss of B cell function. Conditions resulting in anacquired loss of B cell function that may be ameliorated or treated byadministering the polypeptides, antibodies, polynucleotides and/oragonists or antagonists thereof, include, but are not limited to, HIVInfection, AIDS, bone marrow transplant, and B cell chronic lymphocyticleukemia (CLL).

[0485] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to boost immunoresponsiveness among individualshaving a temporary immune deficiency. Conditions resulting in atemporary immune deficiency that may be ameliorated or treated byadministering the polypeptides, antibodies, polynucleotides and/oragonists or antagonists thereof, include, but are not limited to,recovery from viral infections (e.g., influenza), conditions associatedwith malnutrition, recovery from infectious mononucleosis, or conditionsassociated with stress, recovery from measles, recovery from bloodtransfusion, and recovery from surgery.

[0486] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a regulator of antigen presentation by monocytes, dendriticcells, and/or B-cells. In one embodiment, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventionenhance antigen presentation or antagonizes antigen presentation invitro or in vivo. Moreover, in related embodiments, said enhancement orantagonism of antigen presentation may be useful as an anti-tumortreatment or to modulate the immune system.

[0487] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as an agent to direct an individual's immune system towardsdevelopment of a humoral response (i.e. TH2) as opposed to a TH1cellular response.

[0488] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

[0489] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a stimulator of B cell production in pathologies such asAIDS, chronic lymphocyte disorder and/or Common VariableImmunodeficiency.

[0490] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for generation and/or regeneration of lymphoidtissues following surgery, trauma or genetic defect. In another specificembodiment, polypeptides, antibodies, polynucleotides and/or agonists orantagonists of the present invention are used in the pretreatment ofbone marrow samples prior to transplant.

[0491] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a gene-based therapy for genetically inherited disordersresulting in immuno-incompetence/immunodeficiency such as observed amongSCID patients.

[0492] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of activating monocytes/macrophages to defendagainst parasitic diseases that effect monocytes such as Leishmania.

[0493] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of regulating secreted cytokines that are elicitedby polypeptides of the invention.

[0494] In another embodiment, polypeptides, antibodies, polynucleotidesand/or agonists or antagonists of the present invention are used in oneor more of the applications described herein, as they may apply toveterinary medicine.

[0495] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of blocking various aspects of immune responses toforeign agents or self. Examples of diseases or conditions in whichblocking of certain aspects of immune responses may be desired includeautoimmune disorders such as lupus, and arthritis, as well asimmunoresponsiveness to skin allergies, inflammation, bowel disease,injury and diseases/disorders associated with pathogens.

[0496] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for preventing the B cell proliferation and Igsecretion associated with autoimmune diseases such as idiopathicthrombocytopenic purpura, systemic lupus erythematosus and multiplesclerosis.

[0497] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a inhibitor of B and/or T cell migration in endothelialcells. This activity disrupts tissue architecture or cognate responsesand is useful, for example in disrupting immune responses, and blockingsepsis.

[0498] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for chronic hypergammaglobulinemia evident in suchdiseases as monoclonal gammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonal gammopathies, andplasmacytomas.

[0499] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay be employed for instance to inhibit polypeptide chemotaxis andactivation of macrophages and their precursors, and of neutrophils,basophils, B lymphocytes and some T-cell subsets, e.g., activated andCD8 cytotoxic T cells and natural killer cells, in certain autoimmuneand chronic inflammatory and infective diseases. Examples of autoimmunediseases are described herein and include multiple sclerosis, andinsulin-dependent diabetes.

[0500] The polypeptides, antibodies, polynucleotides and/or agonists orantagonists of the present invention may also be employed to treatidiopathic hyper-eosinophilic syndrome by, for example, preventingeosinophil production and migration.

[0501] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used to enhance or inhibit complement mediated cell lysis.

[0502] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used to enhance or inhibit antibody dependent cellular cytotoxicity.

[0503] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay also be employed for treating atherosclerosis, for example, bypreventing monocyte infiltration in the artery wall.

[0504] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay be employed to treat adult respiratory distress syndrome (ARDS).

[0505] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionmay be useful for stimulating wound and tissue repair, stimulatingangiogenesis, and/or stimulating the repair of vascular or lymphaticdiseases or disorders. Additionally, agonists and antagonists of theinvention may be used to stimulate the regeneration of mucosal surfaces.

[0506] In a specific embodiment, polynucleotides or polypeptides, and/oragonists thereof are used to diagnose, prognose, treat, and/or prevent adisorder characterized by primary or acquired immunodeficiency,deficient serum immunoglobulin production, recurrent infections, and/orimmune system dysfunction. Moreover, polynucleotides or polypeptides,and/or agonists thereof may be used to treat or prevent infections ofthe joints, bones, skin, and/or parotid glands, blood-borne infections(e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis),autoimmune diseases (e.g., those disclosed herein), inflammatorydisorders, and malignancies, and/or any disease or disorder or conditionassociated with these infections, diseases, disorders and/ormalignancies) including, but not limited to, CVID, other primary immunedeficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitismedia, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster(e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseasesand disorders that may be prevented, diagnosed, prognosed, and/ortreated with polynucleotides or polypeptides, and/or agonists of thepresent invention include, but are not limited to, HIV infection,HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunctionanemia, thrombocytopenia, and hemoglobinuria.

[0507] In another embodiment, polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention are used totreat, and/or diagnose an individual having common variableimmunodeficiency disease (“CVID”; also known as “acquiredagammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset ofthis disease.

[0508] In a specific embodiment, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention maybe used to diagnose, prognose, prevent, and/or treat cancers orneoplasms including immune cell or immune tissue-related cancers orneoplasms. Examples of cancers or neoplasms that may be prevented,diagnosed, or treated by polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention include, but arenot limited to, acute myelogenous leukemia, chronic myelogenousleukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocyticanemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiplemyeloma, Burkitt's lymphoma, and/or EBV-transformed diseases.

[0509] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a therapy for decreasing cellular proliferation of LargeB-cell Lymphomas.

[0510] In another specific embodiment, polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the present inventionare used as a means of decreasing the involvement of B cells and Igassociated with Chronic Myelogenous Leukemia.

[0511] In specific embodiments, the compositions of the invention areused as an agent to boost immunoresponsiveness among B cellimmunodeficient individuals, such as, for example, an individual who hasundergone a partial or complete splenectomy.

[0512] Antagonists of the invention include, for example, binding and/orinhibitory antibodies, antisense nucleic acids, ribozymes or solubleforms of the polypeptides of the present invention (e.g., Fc fusionprotein). Agonists of the invention include, for example, binding orstimulatory antibodies, and soluble forms of the polypeptides (e.g., Fcfusion proteins), polypeptides, antibodies, polynucleotides and/oragonists or antagonists of the present invention may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., asdescribed herein.

[0513] In another embodiment, polypeptides, antibodies, polynucleotidesand/or agonists or antagonists of the present invention are administeredto an animal (including, but not limited to, those listed above, andalso including transgenic animals) incapable of producing functionalendogenous antibody molecules or having an otherwise compromisedendogenous immune system, but which is capable of producing humanimmunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT Application Nos. WO98/24893, WO/9634096, WO/9633735, andWO/9110741). Administration of polypeptides, antibodies, polynucleotidesand/or agonists or antagonists of the present invention to such animalsis useful for the generation of monoclonal antibodies against thepolypeptides, antibodies, polynucleotides and/or agonists or antagonistsof the present invention.

[0514] Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof are useful in promoting angiogenesisand/or wound healing (e.g., wounds, burns, and bone fractures).

[0515] Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof are also useful as an adjuvant toenhance immune responsiveness. In specific embodiments, thepolynucleotides and or polypeptides of the invention and/or agonistsand/or antagonists thereof are used as an adjuvant to enhance immuneresponsiveness to specific antigens. In particular, polynucleotides andor polypeptides of the invention and/or agonists and/or antagoniststhereof are used as an adjuvant to enhance anti-viral immune responses.

[0516] More generally, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful inregulating (i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, transplantation and burns, or may be used to boost immuneresponse and/or recovery in the elderly and/or immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment, prevention,diagnosis, and/or detection of autoimmune disorders. In specificembodiments, polynucleotides and/or polypeptides of the invention areused to treat, prevent, diagnose, and/or detect chronic inflammatory,allergic or autoimmune conditions, such as those described herein or areotherwise known in the art.

[0517] Since TNF-gamma-alpha and TNF-gamma-beta belong to the TNFsuperfamily, they also modulate angiogenesis. In addition, sinceTNF-gamma-alpha and/or TNF-gamma-beta inhibit immune cell functions, itwill have a wide range of anti-inflammatory activities. TNF-gamma-alphaand/or TNF-gamma-beta may be employed as an anti-neovascularizing agentto treat, prevent, diagnose, and/or detect solid tumors by stimulatingthe invasion and activation of host defense cells, e.g., cytotoxicT-cells and macrophages and by inhibiting the angiogenesis of tumors.Those of skill in the art will recognize other non-cancer indicationswhere blood vessel proliferation is not wanted. They may also beemployed to enhance host defenses against resistant chronic and acuteinfections, for example, myobacterial infections via the attraction andactivation of microbicidal leukocytes. TNF-gamma-alpha and/orTNF-gamma-beta may also be employed to inhibit T-cell proliferation bythe inhibition of IL-2 biosynthesis for the treatment, prevention,diagnosis, and/or detection of T-cell mediated auto-immune diseases andlymphocytic leukemias. TNF-gamma-alpha and/or TNF-gamma-beta may also beemployed to stimulate wound healing, both via the recruitment of debrisclearing- and connective tissue promoting- inflammatory cells. In thissame manner, TNF-gamma-alpha and/or TNF-gamma-beta may also be employedto treat, prevent, diagnose, and/or detect other fibrotic disorders,including liver cirrhosis, osteoarthritis and pulmonary fibrosis.TNF-gamma-alpha and/or TNF-gamma-beta also increases the presence ofeosinophils which have the distinctive function of killing the larvae ofparasites that invade tissues, as in schistosomiasis, trichinosis andascariasis. TNF-gamma-alpha and/or TNF-gamma-beta may also be employedto regulate hematopoiesis, by regulating the activation anddifferentiation of various hematopoietic progenitor cells, for example,to release mature leukocytes from the bone marrow followingchemotherapy, i.e., in stem cell mobilization. TNF-gamma-alpha and/orTNF-gamma-beta may also be employed to treat, prevent, diagnose, and/ordetect sepsis.

[0518] It is well-known in the art that, in addition to a specificcellular function, cellular receptor molecules may also often beexploited by a virus as a means of initiating entry into a potentialhost cell. For example, it was recently discovered by Wu and colleagues(J. Exp. Med. 185:1681-1691 (1997)) that the cellular chemokine receptorCCR5 functions not only as a cellular chemokine receptor, but also as areceptor for macrophage-tropic human immunodeficiency virus (HIV)-1. Inaddition, RANTES, MIP-1alpha, and MIP-1beta, which are agonists for thecellular chemokine receptor CCR5, inhibit entry of various strains ofHIV-1 into susceptible cell lines (Cocchi, F., et al., Science270:1811-1815 (1995)). Thus, the invention also provides a method oftreating, preventing, diagnosing, and/or detecting an individual exposedto, or infected with, a virus through the prophylactic or therapeuticadministration of TNF-gamma-alpha and/or TNF-gamma-beta, or an agonistor antagonist thereof, to block or disrupt the interaction of a viralparticle with the TNF-gamma-alpha and/or TNF-gamma-beta receptor and, asa result, block the initiation or continuation of viral infectivity.

[0519] The TNF-gamma-alpha and/or TNF-gamma-beta of the presentinvention binds to the TNF-gamma-alpha and/or TNF-gamma-beta receptorand, as such, is likely to block immuno- and endothelial cell-tropicviral infections. Expression patterns of the cDNA clone encoding thepresent invention suggests that this molecule is expressed primarily inendothelial cells and select hematopoietic tissues. When consideredtogether, these observations suggest that agonists and antagonists,including a receptor, of TNF-gamma-alpha and/or TNF-gamma-beta may beuseful as a method of blocking or otherwise regulating the infectivityof immunotropic viral infections. A non-limiting list of viruses whichinfect humans and can infect cells of the hematopoietic system includessuch retroviruses as HIV-1, HIV-2, human T-cell lymphotropic virus(HTLV)-I, and HTLV-II, as well as other DNA and RNA viruses such asherpes simplex virus (HSV)-1, HSV-2, HSV-6, cytomegalovirus (CMV),Epstein-Barr virus (EBV), herpes samirii, adenoviruses, rhinoviruses,influenza viruses, reoviruses, and the like.

[0520] The ability of TNF-gamma-alpha and/or TNF-gamma-beta of thepresent invention, or agonists or antagonists thereof, toprophylactically or therapeutically block viral infection may be easilytested by the skilled artisan. For example, Simmons and coworkers(Science 276:276-279 (1997)) and Arenzana-Seisdedos and colleagues(Nature 383:400 (1996)) each outline a method of observing suppressionof HIV-1 infection by an antagonist of the CCR5 chemokine receptor andof the CC chemokine RANTES, respectively, in cultured peripheral bloodmononuclear cells. Cells are cultured and infected with a virus, HIV-1in both cases noted above. An agonist or antagonist of the CC chemokineor its receptor is then immediately added to the culture medium.Evidence of the ability of the agonist or antagonist of the chemokine orcellular receptor is determined by evaluating the relative success ofviral infection at 3, 6, and 9 days postinfection.

[0521] Administration of a pharmaceutical composition comprising anamount of an isolated TNF-gamma-alpha and/or TNF-gamma-beta, or anagonist or antagonist thereof, of the invention to an individual eitherinfected with a virus or at risk for infection with a virus is performedas described below.

[0522] Since TNF-gamma has been shown to induce activation of cellularNF-KB and c-jun N-terminal kinase (JNK), it is also useful intherapeutically regulating such cellular and immune systemic disordersas tumors and tumor metastases, infections by bacteria, viruses, andother parasites, immunodeficiencies, inflammatory diseases,lymphadenopathy, autoimmune diseases, graft versus host disease,autoimmunity, arthritis, leukemias, lymphomas, immunosuppression,inflammatory bowel disease, myelosuppression, and related sequelae.

[0523] The present invention is also useful for treatment, prevention,diagnosis, and/or detection of various immune and circulatorysystem-related disorders in mammals, preferably humans. Such disordersinclude tumors (a nonlimiting list of human tumors includes breastcancer, colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, adenoma, and the like) andtumor metastasis, infections by bacteria, viruses, and other parasites,immunodeficiencies, inflammatory diseases, lymphadenopathy, autoimmunediseases, graft versus host disease, and any disregulation of immune andcirculatory systems cell function including, but not limited to,autoimmunity, arthritis, leukemias, lymphomas, immunosuppression,immunity, humoral immunity, inflammatory bowel disease, myelosuppression, and the like.

[0524] TNF-gamma-alpha and/or TNF-gamma-beta polypeptides orpolynucleotides encoding TNF-gamma-alpha and/or TNF-gamma-beta of theinvention (including TNF-gamma-alpha and/or TNF-gamma-beta agonists orantagonists) may be used to treat, prevent, diagnose, and/or detectcardiovascular disorders, including peripheral artery disease, such aslimb ischemia.

[0525] Cardiovascular disorders include cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisenmenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

[0526] Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[0527] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0528] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[0529] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0530] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0531] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[0532] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0533] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0534] Cerebrovascular disorders include carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[0535] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromboembolisms. Thrombosis include coronary thrombosis,hepatic vein thrombosis, retinal vein occlusion, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[0536] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[0537] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail.

[0538] Unregulated angiogenesis becomes pathologic and sustainsprogression of many neoplastic and non-neoplastic diseases. A number ofserious diseases are dominated by abnormal neovascularization includingsolid tumor growth and metastases, arthritis, some types of eyedisorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech.9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763(1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman,Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press,New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982);and Folkman et al., Science 221:719-725 (1983). In a number ofpathological conditions, the process of angiogenesis contributes to thedisease state. For example, significant data have accumulated whichsuggest that the growth of solid tumors is dependent on angiogenesis.Folkman and Klagsbrun, Science 235:442-447 (1987).

[0539] The present invention provides for treatment, prevention,diagnosis, and/or detection of diseases or disorders associated withneovascularization by administration of the TNF-gamma-alpha and/orTNF-gamma-beta polynucleotides and/or polypeptides of the invention(including TNF-gamma-alpha and/or TNF-gamma-beta agonists and/orantagonists). Malignant and metastatic conditions which can be treated,prevented, diagnosed, and/or detected with the polynucleotides andpolypeptides of the invention include, but are not limited to thosemalignancies, solid tumors, and cancers described herein and otherwiseknown in the art (for a review of such disorders, see Fishman et al.,Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)):

[0540] Additionally, ocular disorders associated with neovascularizationwhich can be treated, prevented, diagnosed, and/or detected with theTNF-gamma-alpha and/or TNF-gamma-beta polynucleotides and polypeptidesof the present invention (including TNF-gamma-alpha and/orTNF-gamma-beta agonists and TNF-gamma-alpha and/or TNF-gamma-betaantagonists) include, but are not limited to: neovascular glaucoma,diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis,retinopathy of prematurity macular degeneration, corneal graftneovascularization, as well as other eye inflammatory diseases, oculartumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

[0541] Additionally, disorders which can be treated, prevented,diagnosed, and/or detected with the TNF-gamma-alpha and/orTNF-gamma-beta polynucleotides and polypeptides of the present invention(including TNF-gamma-alpha and/or TNF-gamma-beta agonists andTNF-gamma-alpha and/or TNF-gamma-beta antagonists) include, but are notlimited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

[0542] In a similar fashion, TNF-gamma-alpha and/or TNF-gamma-beta maybe used to treat, prevent, diagnose, and/or detect rheumatoid arthritis(RA) by inhibiting the increase in angiogensis or the increase inendothelial cell proliferation required to sustain an invading pannus inbone and cartilage as is often observed in RA. Endothelial cellproliferation is increased in the synovia of RA patients as compared topatients with osteoarthritis (OA) or unaffected individuals.Neovascularization is needed to sustain the increased mass of theinvading pannus into bone and cartilage. Inhibition of angiogenesis isassociated with a significant decrease in the severity of both early andchronic arthritis in animal models.

[0543] The TNF-gamma-alpha and/or TNF-gamma-beta polypeptide of thepresent invention may be employed to inhibit tumor cell growth orneoplasia. The TNF-gamma-alpha and/or TNF-gamma-beta polypeptide may beresponsible for tumor destruction through apoptosis which ischaracterized by membrane blebbing (zeiosis), condensation of cytoplasmaand the activation of an endogenous endonuclease (FIG. 12). As shown inTable 1, TNF-gamma has strong cytotoxic activity for the cell linestested which have abnormal cellular proliferation and regulation, forexample the fibrosarcoma and carcinoma cell line. This is alsoillustrated in FIGS. 7A, 7B, and 8 where it is shown that TNF-gamma hasthe ability to inhibit L929 and WEHI 164 cell growth through cytotoxicactivity. WEHI 164 cells are mouse fibrosarcoma cells. A preferablemethod of administering the TNF-gamma is by injection directly into thetumor.

[0544] Diseases or conditions that may be treated, prevented, diagnosed,and/or detected with the polynucleotides or polypeptides of theinvention include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

[0545] The cell adhesion activity of TNF-gamma may be employed for woundhealing. As shown in Table 1 and FIG. 9, TNF-gamma has a strongendothelial cell proliferation effect which is an indication thatTNF-gamma plays a role in wound healing. TNF-gamma's cell adhesiveeffects may also play a role in wound healing.

[0546] TNF-gamma may also be employed to treat, prevent, diagnose,and/or detect diseases which require growth promotion activity, forexample, restenosis. As stated above, TNF-gamma is shown to have strongproliferation effects on endothelial cell growth. Accordingly, TNF-gammamay also be employed to regulate hematopoiesis and endothelial celldevelopment.

[0547] The TNF-gamma polypeptide, through its ability to stimulate theactivation of T-cells, is an important mediator of the immune response.Accordingly, this polypeptide may be used to stimulate an immuneresponse against a variety of parasitic, bacterial and viral infections.TNF-gamma may lyse virus-infected cells and, therefore, be employed toarrest HIV infected cells.

[0548] The TNF-gamma polypeptide may also be employed to treat, prevent,diagnose, and/or detect autoimmune diseases such as Type I diabetes byenhancing the T-cell proliferative response. TABLE 2 Summary ofTNF-gamma activity Cyto- Source tox- Prolif- Differen- Cell lines andtype icity eration tiation Adhesion L929 mouse + − − − fibroblast WEHI164 mouse +++ − − − fibrosarcoma NRK-54E rat kidney + − − −epithelial-like THP-1 human + − ++ ++ monocytic leukemia HL-60 human − −− ++ promyelocytic leukemia Raji human − − − − Burkitt lymphoma Jurkathuman ++ − − − T-cell lymphoma Primary HUVEC − ++ − ? Primary humanaterial +* ++ − ? endothelial A-431 human ++ − − − epidermoid carcinoma293 human − ++ − − embryonal

[0549] Legend: * At high dose only. The numbers of “+” indicate therelative level of activities. “−” indicates no detected activity at theconcentration range tested.

[0550] TNF-gamma may be used to inhibit the proliferation of endothelialcells, for example, aortic endothelial cells. As illustrated in FIG. 10,at concentrations of up to 10 μg/ml, TNF-gamma can nearly completelyinhibit the growth of endothelial cells while having no apparent effecton the growth of human breast cancer cells. As a result, TNF-gamma canbe used to treat, prevent, diagnose, and/or detect diseases anddisorders in which inhibition of endothelial cell growth isadvantageous. Inhibiting the growth of endothelial cells is desirable inthe treatment of many types of cancers which depend on the generation ofnew blood vessels to support growth of the tumor. TNF-gamma can be usedto inhibit the growth of such tumors by inhibiting the growth ofendothelial cells which are a major cellular component of the bloodvessel. Evidence of the ability of TNF-gamma to be effectively used inthis fashion is presented in FIGS. 16A and 16B. These experiments arediscussed in greater detail below.

[0551] In particular, TNF-gamma can be used to regulate endothelial cellgrowth when endothelial cells have already begun proliferating. Such asituation may arise when angiogenesis is occurring as a tumor-supportingmechanism as described above. Endogenous TNF-gamma expression is reducedin proliferating cultures of endothelial cells, whereas the expressionof endogenous TNF-gamma is enhanced in quiescent endothelial cellcultures (FIG. 4). As a result, it is preferable to use TNF-gamma of thepresent invention to reduce the rate of cell growth in cultures ofproliferating endothelial cells, for example, during the increase insize of a tumor in a cancerous state.

[0552] TNF-gamma of the present invention has been used to reduce theformation of capillary-like tubular structures formed by endothelialcells in vitro. As illustrated in FIG. 14, TNF-gamma of the presentinvention can be used to inhibit the formation of endothelial cellsorganized into capillary-like tubular structures in response toangiogenic factors such as FGF-2. Furthermore, isolated TNF-gamma of thepresent invention can also be used to inhibit the growth andorganization of endothelial cells into capillary vessels in a modifiedchicken embryo chorioallantoic membrane (CAM), as shown in FIG. 15. As aresult, TNF-gamma of the present invention can be used to inhibit theformation of capillaries or capillary-like structures from endothelialcells in vitro.

[0553] TNF-gamma of the present invention can be used as an anti-canceragent. As illustrated in FIG. 16, TNF-gamma was used to inhibit thegrowth of human breast cancer cells in a xenograft tumor model. Despitethe high tumorigenicity of these cells, treatment with TNF-gamma of thepresent invention resulted in a marked inhibition of the growth of thexenograft tumors. TNF-gamma, or a mutein thereof, of the presentinvention, can be used to treat, prevent, diagnose, and/or detect anumber of cancers including, but not limited to, breast cancer, coloncancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, adenoma, and the like.

[0554] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatment, prevention, diagnosis, and/or detection of human disease.

[0555] This invention provides a method for identification of thereceptor for TNF-gamma. The gene encoding the receptor can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting (Coligan, et al., Current Protocols inImmun., 1(2), Chapter 5, (1991)). Preferably, expression cloning isemployed wherein polyadenylated RNA is prepared from a cell responsiveto TNF-gamma, and a cDNA library created from this RNA is divided intopools and used to transfect COS cells or other cells that are notresponsive to TNF-gamma. Transfected cells which are grown on glassslides are exposed to labeled TNF-gamma. TNF-gamma can be labeled by avariety of means including iodination or inclusion of a recognition sitefor a site-specific protein kinase. Following fixation and incubation,the slides are subjected to autoradiographic analysis. Positive poolsare identified and sub-pools are prepared and retransfected using aniterative sub-pooling and rescreening process, eventually yielding asingle clone that encodes the putative receptor.

[0556] As an alternative approach for receptor identification, labeledTNF-gamma can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the TNF-gamma-receptor can be excised, resolved into peptidefragments, and subjected to protein microsequencing. The amino acidsequence obtained from microsequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

[0557] TNF-gamma does not bind significantly to two soluble TNFreceptors, sTNF-RI (p55) and sTNF-RII (p75). Accordingly, TNF-gamma mayhave activities inclusive of and additional to known TNF proteins (seeFIG. 13).

Formulations and Administration

[0558] The TNF-gamma polypeptide composition will be formulated anddosed in a fashion consistent with good medical practice, taking intoaccount the clinical condition of the individual patient (especially theside effects of treatment with TNF-gamma polypeptide alone), the site ofdelivery of the TNF-gamma polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of TNF-gamma polypeptidefor purposes herein is thus determined by such considerations.

[0559] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0560] The TNF-gamma polypeptides and agonists and antagonists of thepresent invention may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the compound, and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

[0561] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions of the present invention maybe employed in conjunction with other therapeutic compounds.

[0562] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10micrograms/kg body weight and in most cases they will be administered inan amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 micrograms/kg to about 1 mg/kg bodyweight daily, taking into account the routes of administration,symptoms, etc.

[0563] The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, growth factors, steroidal andnon-steroidal anti-inflammatories, conventional immunotherapeuticagents, cytokines, chemokines, growth factors, radiotherapy and/or orradiation therapy. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second. In certain embodiments, compositions of theinvention are administered in combination with one or more othertherapeutic agents (including, for example, a one or morechemotherapeutic agents and/or radiotherapy and/or radiation therapy),wherein either or both the compositions of the invention and/or one ormore of the therapeutic agents are administered at standard, reduced orincreased dosages.

[0564] In one embodiment, the compositions of the invention areadministered in combination with other members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with thecompositions of the invention include, but are not limited to, solubleforms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known asTNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL,FasL, CD27L, CD30L, CD40L, 4-IBBL, DcR3, OX40L, AIM-I (InternationalPublication No. WO 97/33899), AIM-II (International Publication No. WO97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha(International Publication No. WO 98/07880), TR6 (InternationalPublication No. WO 98/30694), OPG, and neutrokine-alpha (InternationalPublication No. WO 98/18921, OX40, and nerve growth factor (NGF), andsoluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (InternationalPublication No. WO 96/34095), DR3 (International Publication No. WO97/33904), DR4 (International Publication No. WO 98/32856), TR5(International Publication No. WO 98/30693), TR6 (InternationalPublication No. WO 98/30694), TR7 (International Publication No. WO98/41629), TRANK, TR9 (International Publication No. WO 98/56892),TR10(International Publication No. WO 98/54202), 312C2 (InternationalPublication No. WO 98/06842), and TR12, and soluble forms CD154, CD70,and CD153.

[0565] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0566] In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,beta-lactamases, aminoglycosides, macrolides, quinolones,fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, andstreptomycin.

[0567] In certain embodiments, compositions of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddl), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat AIDS and/or to prevent ortreat HIV infection.

[0568] In other embodiments, compositions of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, compositions of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, compositions of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, compositions of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, compositions of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, compositions of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

[0569] In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[0570] In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

[0571] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0572] Additionally, immunosuppressants preparations that may beadministered with the compositions of the invention include, but are notlimited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

[0573] In a preferred embodiment, the compositions of the invention areadministered in combination with steroid therapy. Steroids that may beadministered in combination with the compositions of the invention,include, but are not limited to, oral corticosteroids, prednisone, andmethylprednisolone (e.g., IV methylprednisolone). In a specificembodiment, compositions of the invention are administered incombination with prednisone. In a further specific embodiment, thecompositions of the invention are administered in combination withprednisone and an immunosuppressive agent. Immunosuppressive agents thatmay be administered with the compositions of the invention andprednisone are those described herein, and include, but are not limitedto, azathioprine, cylophosphamide, and cyclophosphamide IV. In a anotherspecific embodiment, compositions of the invention are administered incombination with methylprednisolone. In a further specific embodiment,the compositions of the invention are administered in combination withmethylprednisolone and an immunosuppressive agent. Immunosuppressiveagents that may be administered with the compositions of the inventionand methylprednisolone are those described herein, and include, but arenot limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.

[0574] In a preferred embodiment, the compositions of the invention areadministered in combination with an antimalarial. Antimalarials that maybe administered with the compositions of the invention include, but arenot limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

[0575] In a preferred embodiment, the compositions of the invention areadministered in combination with an NSAID.

[0576] In a nonexclusive embodiment, the compositions of the inventionare administered in combination with one, two, three, four, five, ten,or more of the following drugs: NRD-101 (Hoechst Marion Roussel),diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin(Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton(Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470(Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000(Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra genetherapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech),DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), soluble TNFreceptor 1 (synergen; Amgen), IPR-6001 (Institute for PharmaceuticalResearch), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals),BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim),LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), butixocortpropionate (WarnerLambert).

[0577] In a preferred embodiment, the compositions of the invention areadministered in combination with carboplatin and paclitaxel or withcisplatin and etoposide.

[0578] In a preferred embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five or more ofthe following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,auranofin, cyclosporine, penicillamine, azathioprine, an antimalarialdrug (e.g., as described herein), cyclophosphamide, chlorambucil, gold,ENBREL™ (Etanercept), anti-TNF antibody, and prednisolone.

[0579] In a more preferred embodiment, the compositions of the inventionare administered in combination with an antimalarial, methotrexate,anti-TNF antibody, ENBREL™ and/or suflasalazine. In one embodiment, thecompositions of the invention are administered in combination withmethotrexate. In another embodiment, the compositions of the inventionare administered in combination with anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with methotrexate and anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with suflasalazine. In another specific embodiment, thecompositions of the invention are administered in combination withmethotrexate, anti-TNF antibody, and suflasalazine. In anotherembodiment, the compositions of the invention are administered incombination ENBREL™. In another embodiment, the compositions of theinvention are administered in combination with ENBREL™ and methotrexate.In another embodiment, the compositions of the invention areadministered in combination with ENBREL™, methotrexate andsuflasalazine. In another embodiment, the compositions of the inventionare administered in combination with ENBREL™, methotrexate andsuflasalazine. In other embodiments, one or more antimalarials iscombined with one of the above-recited combinations. In a specificembodiment, the compositions of the invention are administered incombination with an antimalarial (e.g., hydroxychloroquine), ENBREL™,methotrexate and suflasalazine. In another specific embodiment, thecompositions of the invention are administered in combination with anantimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNFantibody, and methotrexate.

[0580] In an additional embodiment, compositions of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the compositions of the invention include, but notlimited to, GAMMAR™, IVEEGAMN™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

[0581] CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND™),biologically active fragments, variants, or derivatives of CD40L,anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies),and/or anti-CD40 antibodies (e.g., agonistic or antagonisticantibodies).

[0582] In an additional embodiment, the compositions of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with thecompositions of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[0583] In another embodiment, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the compositionsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0584] In an additional embodiment, the compositions of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha.

[0585] In one embodiment, the compositions of the invention areadministered in combination with one or more chemokines. In specificembodiments, the compositions of the invention are administered incombination with an alpha (CxC) chemokine selected from the groupconsisting of alpha interferon inducible protein-10 (IP-10),interleukin-8 (IL-8), platelet factor-4 (PF4), neutrophil activatingprotein (NAP-2), GRO-alpha, GRO-alpha, GRO-alpha, neutrophil-activatingpeptide (ENA-78), granulocyte chemoattractant protein-2 (GCP-2), andstromal cell-derived factor-1 (SDF-1, or pre-B cell stimulatory factor(PBSF)); and/or an alpha (CC) chemokine selected from the groupconsisting of: RANTES (regulated on activation, normal T expressed andsecreted), macrophage inflammatory protein-1 alpha (MIP-1 alpha),macrophage inflammatory protein-1 alpha (MIP-1 alpha), monocytechemotactic protein-1 (MCP-1), monocyte chemotactic protein-2 (MCP-2),monocyte chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4(MCP-4) macrophage inflammatory protein-1 alpha (MIP-1 alpha),macrophage inflammatory protein-3alpha (MIP-3 alpha), macrophageinflammatory protein-3 alpha (MIP-3 alpha), macrophage inflammatoryprotein-4 (MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and I-309; and/or thealpha (C) chemokine, lymphotactin.

[0586] In an additional embodiment, the compositions of the invention(e.g., antagonists) are administered in combination with angiogenicproteins or compounds. Angiogenic proteins that may be administered withthe compositions of the invention include, but are not limited to,Glioma Derived Growth Factor (GDGF), as disclosed in European Pat.Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), asdisclosed in European Pat. Number EP-682110; Platelet Derived GrowthFactor-B (PDGF-B), as disclosed in European Pat. Number EP-282317;Placental Growth Factor (PIGF), as disclosed in InternationalPublication Number WO 92/06194; Placental Growth Factor-2 (PIGF-2), asdisclosed in Hauser et al., Gorwth Factors, 4:259-268 (1993); VascularEndothelial Growth Factor (VEGF), as disclosed in InternationalPublication Number WO 90/13649; Vascular Endothelial Growth Factor-A(VEGF-A), as disclosed in European Pat. Number EP-506477; VascularEndothelial Growth Factor-2 (VEGF-2), as disclosed in InternationalPublication Number WO 96/39515; Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Pat. Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

[0587] In additional embodiments, the compositions of the invention areadministered in combination with anti-angiogenic proteins or compoundsand/or antagonists thereof.

[0588] In additional embodiments, the compositions of the invention areadministered, either alone or in combination with one or more additionalagents or compounds (as described herein), in a dose-cycling fashion.For example, a composition of the invention may be administered inrepeatedly increasing and decreasing doses either alone, in unison withone or more additional agents or compounds, or in a complementarydose-cycling fashion with one or more additional agents or compounds(such that the dose of the composition of the invention is relativelyhigh in concert with a relatively low dose of one or more additionalagents or compounds and vice versa). In a preferred embodiment,dose-cycling with one or more compositions of the inventionadministered, either alone or in combination with one or more additionalagents or compounds, is used to treat tumors. In another preferredembodiment, dose-cycling with one or more compositions of the inventionadministered, either alone or in combination with one or more additionalagents or compounds, is used to inhibit angiogenesis (either in part orin full). In a highly preferred embodiment, dose-cycling with one ormore compositions of the invention administered, either alone or incombination with one or more additional agents or compounds, is used toinhibit angiogenesis (either in part or in whole) and to thereby treat atumor.

[0589] In an additional embodiment, the compositions of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors tha may be administered with the compositionsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[0590] In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Gene Therapy

[0591] The TNF-gamma polypeptides and agonists and antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

[0592] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral particle containing RNA encoding a polypeptide ofthe present invention.

[0593] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a producer cell for producing a retroviral particle containingRNA encoding a polypeptide of the present invention may be administeredto a patient for engineering cells in vivo and expression of thepolypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0594] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0595] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed by Miller and colleagues (Biotechniques 7:980-990 (1989)), orany other promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andb-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

[0596] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAl promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0597] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, b-2, b-AM, PA12, T19-14X, VT-19-17-H2, CRE, b-CRIP, GP+E-86,GP+envAm12, and DAN cell lines as described by Miller (Human GeneTherapy 1:5-14 (1990)), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0598] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Agonists and Antagonists—Assays and Molecules

[0599] This invention is also related to a method of screening compoundsto identify those which mimic TNF-gamma (agonists) or prevent the effectof TNF-gamma (antagonists). An example of such a method takes advantageof the ability of TNF-gamma to significantly stimulate the proliferationof human endothelial cells in the presence of the comitogen Con A.Endothelial cells are obtained and cultured in 96-well flat-bottomedculture plates (Costar, Cambridge, Mass.) in RPM1 1640 supplemented with10% heat-inactivated fetal bovine serum (Hyclone Labs, Logan, Utah), 1%L-glutamine, 100 U/ml penicillin, 100 micrograms/ml streptomycin, 0.1%gentamycin (Gibco Life Technologies, Grand Island, N.Y.) in the presenceof 2 micrograms/ml of Con-A (Calbiochem, La Jolla, Calif.). Con-A, andthe compound to be screened are added to a final volume of 0.2 ml. After60 h at 37° C., cultures are pulsed with 1 microCi of [³H]thymidine (5Ci/mmol; 1 Ci=37 BGq; NEN) for 12-18 h and harvested onto glass fiberfilters (PhD; Cambridge Technology, Watertown, Mass.). Mean[³H]thymidine incorporation (cpm) of triplicate cultures is determinedusing a liquid scintillation counter (Beckman Instruments, Irvine,Calif.). Significant [³H]-thymidine incorporation indicates stimulationof endothelial cell proliferation.

[0600] Alternatively, the response of a known second messenger systemfollowing interaction of TNF-gamma and receptor would be measured andcompared in the presence or absence of the compound. Such secondmessenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

[0601] To assay for antagonists, the assay described above is performed,however, in this assay TNF-gamma is added along with the compound to bescreened and the ability of the compound to inhibit [³H]thymidineincorporation in the presence of TNF-gamma, indicates that the compoundis an antagonist to TNF-gamma. Alternatively, TNF-gamma antagonists maybe detected by combining TNF-gamma and a potential antagonist withmembrane-bound TNF-gamma receptors or recombinant receptors underappropriate conditions for a competitive inhibition assay. TNF-gamma canbe labeled, such as by radioactivity, such that the number of TNF-gammamolecules bound to the receptor can determine the effectiveness of thepotential antagonist.

[0602] Alternatively, a mammalian cell or membrane preparationexpressing the TNF-gamma receptor is incubated with labeled TNF-gamma inthe presence of the compound. The ability of the compound to enhance orblock this interaction could then be measured.

[0603] In another aspect of this embodiment the invention provides amethod for identifying a receptor protein or other ligand-bindingprotein which binds specifically to a TNF-gamma polypeptide (e.g. DR3).For example, a cellular compartment, such as a membrane or a preparationthereof, may be prepared from a cell that expresses a molecule thatbinds TNF-gamma. The preparation is incubated with labeled TNF-gamma andcomplexes of TNF-gamma bound to the receptor or other binding proteinare isolated and characterized according to routine methods known in theart. Alternatively, the TNF-gamma polypeptide may be bound to a solidsupport so that binding molecules solubilized from cells are bound tothe column and then eluted and characterized according to routinemethods.

[0604] In the assay of the invention for agonists or antagonists, acellular compartment, such as a membrane or a preparation thereof, maybe prepared from a cell that expresses a molecule that binds TNF-gamma,such as a molecule of a signaling or regulatory pathway modulated byTNF-gamma. The preparation is incubated with labeled TNF-gamma in theabsence or the presence of a candidate molecule which may be a TNF-gammaagonist or antagonist. The ability of the candidate molecule to bind thebinding molecule is reflected in decreased binding of the labeledligand. Molecules which bind gratuitously, i.e., without inducing theeffects of TNF-gamma on binding the TNF-gamma binding molecule, are mostlikely to be good antagonists. Molecules that bind well and eliciteffects that are the same as or closely related to TNF-gamma areagonists.

[0605] TNF-gamma-like effects of potential agonists and antagonists mayby measured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofTNF-gamma or molecules that elicit the same effects as TNF-gamma. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

[0606] Another example of an assay for TNF-gamma antagonists is acompetitive assay that combines TNF-gamma and a potential antagonistwith membrane-bound TNF-gamma receptor molecules or recombinantTNF-gamma receptor molecules under appropriate conditions for acompetitive inhibition assay. TNF-gamma can be labeled, such as byradioactivity, such that the number of TNF-gamma molecules bound to areceptor molecule can be determined accurately to assess theeffectiveness of the potential antagonist.

[0607] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingTNF-gamma-induced activities, thereby preventing the action of TNF-gammaby excluding TNF-gamma from binding.

[0608] Other potential antagonists include antisense molecules.Antisense technology can be used to control gene expression throughantisense DNA or RNA or through triple-helix formation. Antisensetechniques are discussed in a number of studies (for example, Okano, J.Neurochem. 56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression.” CRC Press, Boca Raton, Fla. (1988)). Triple helixformation is discussed in a number of studies, as well (for instance,Lee, et al., Nucleic Acids Research 6:3073 (1979); Cooney, et al.,Science 241:456 (1988); Dervan, et al., Science 251:1360 (1991)). Themethods are based on binding of a polynucleotide to a complementary DNAor RNA. For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of TNF-gamma. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into TNF-gamma polypeptide. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of TNF-gammaprotein.

[0609] Antibodies specific to TNF-gamma may be used as antagonists bybinding to TNF-gamma and preventing it from binding to its receptor.Monoclonal antibodies are particularly effective in this regard.Antibodies specific to the TNF-gamma receptor, however, may mediatedistinct cellular responses which tend to agonize the effects ofTNF-gamma upon interaction with its receptor.

[0610] Potential TNF-gamma antagonists also include TNF-gamma mutantswhich bind to the TNF-gamma receptor and elicit no second messengerresponse to effectively block the receptor from its natural ligand.Specifically designed oligonucleotides and small molecules may also bindto the TNF-gamma receptor (e.g., DR3) and block it from TNF-gamma.Examples of small molecules include but are not limited to smallpeptides or peptide-like molecules.

[0611] Another potential TNF-gamma antagonist is a soluble form of theTNF-gamma receptor which binds to TNF-gamma and prevents it frominteracting with membrane-bound TNF-gamma receptors. In this way, thereceptors are not stimulated by TNF-gamma.

[0612] Another potential TNF-gamma antagonist is an antisense constructprepared using antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptides of the presentinvention, is used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcription(triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney etal, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360(1991)), thereby preventing transcription and the production ofTNF-gamma. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into the TNF-gammapolypeptide (Antisense—Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of TNF-gamma.

[0613] TNF-alpha antagonists may also be employed to treat, prevent,diagnose, and/or detect cachexia which is a lipid clearing defectresulting from a systemic deficiency of lipoprotein lipase which issuppressed by TNF-gamma. The TNF-gamma antagonists are also employed totreat, prevent, diagnose, and/or detect cerebral malaria in whichTNF-gamma appears to play a pathogenic role. The antagonists may also beemployed to treat, prevent, diagnose, and/or detect rheumatoid arthritisby inhibiting TNF-gamma induced production of inflammatory cytokinessuch as IL-I in the synovial cells. When treating and/or preventingarthritis TNF-gamma is preferably injected intra-articularly.

[0614] The TNF-gamma antagonists may also be employed to prevent graftrejection by preventing the stimulation of the immune system in thepresence of a graft by TNF-gamma.

[0615] The TNF-gamma antagonists may also be employed to treat, prevent,diagnose, and/or detect osteoporosis since TNF-gamma may induce boneresorption.

[0616] Antagonists to TNF-gamma may also be employed asanti-inflammation agents since TNF-gamma mediates an enhancedinflammatory response.

[0617] The antagonists may also be used to treat, prevent, diagnose,and/or detect endotoxic shock, also referred to as septic shock. Thiscritical condition results from an exaggerated response to bacterial orother types of infection. This response leads to elevated levels ofTNF-gamma which causes shock and tissue injury.

[0618] The present invention also relates to a diagnostic assay fordetecting altered levels of TNF-gamma protein in various tissues sincean over-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, tumors and cerebral malaria. Assays used to detectlevels of TNF-gamma protein in a sample derived from a host arewell-known to those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis, ELISA assays and“sandwich” assay. An ELISA assay (Coligan, et al., Current Protocols inImmunology, 1(2), Chapter 6, (1991)) initially comprises preparing anantibody specific to the TNF-gamma antigen, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, flourescence or in this example ahorseradish peroxidase enzyme. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein like BSA. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any TNF-gamma proteins attached to thepolystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to TNF-gamma. Unattached reporter antibody isthen washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of TNF-gamma protein present in a given volume of patientsample when compared against a standard curve.

[0619] A competition assay may be employed wherein antibodies specificto TNF-gamma are attached to a solid support and labeled TNF-gamma and asample derived from the host are passed over the solid support and theamount of label detected, for example by liquid scintillationchromatography, can be correlated to a quantity of TNF-gamma in thesample.

[0620] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay TNF-gamma is passed over a solid support and binds to antibodyattached to a solid support. A second antibody is then bound to theTNF-gamma. A third antibody which is labeled and specific to the secondantibody is then passed over the solid support and binds to the secondantibody and an amount can then be quantitated.

[0621] All of the applications of TNF-gamma described, whether or notexplicitly described herein, also apply to veterinary medicine (in thecontext of, for example, mice, rats, rabbits, hamsters, guinea pigs,pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g.,baboons, monkeys, and chimpanzees).

Gene Mapping

[0622] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0623] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the sequence is used to rapidly select primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

[0624] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0625] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0626] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0627] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0628] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0629] Utilizing the techniques described above, the chromosomallocation of TNF-gamma was determined with very high confidence to be9q32. Previous chromosomal mapping studies have linked severaldevelopmental defects to loci in this area of chromosome 9. In addition,bladder and esophageal cancers have also been associated withchromosomal abnormalities at this locus. See, e.g., Habuchi, T., et al.,Genomics 48:277-88 (1998); Habuchi, T., et al., Hum. Molec. Genet.6:913-19 (1997); Nishiyama, H., et al., Genes Chromosomes Cancer26:171-75 (1999); Miura, K., et al., Cancer Res. 56:1629-34 (1996); andNishiwaki, T., et al., Genes Chromosomes Cancer 27:169-76 (2000).

EXAMPLES

[0630] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0631] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0632] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures, unless otherwise stated. In addition, equivalentplasmids to those described are known in the art and will be apparent tothe ordinarily skilled artisan.

[0633] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0634] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0635] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0636] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0637] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

Example 1 Bacterial Expression and Purification of TNF-gamma

[0638] The DNA sequence encoding the full-length TNF-gamma ORF, ATCCDeposit No. 75927, was initially amplified using PCR oligonucleotideprimers corresponding to the 5′ and 3′ sequences of the TNF-gammaprotein. Additional nucleotides corresponding to TNF-gamma were added tothe 5′ and 3′ sequences respectively. The 5′ oligonucleotide primer isshown as SEQ ID NO:13 and has the sequence 5′-GCG CGG ATC CAC CAT GAGACG CTT TTT AAG CAA AGT C-3′ which contains a Bam HI restriction enzymesite followed by the first 24 nucleotides of TNF-gamma coding sequencestarting from the initiating methionine codon. The 3′ sequence 5′-CGCGTC TAG ACT ATA GTA AGA AGG CTC CAA AGA AGG-3′ (SEQ ID NO:14) containssequences complementary to an Xba I site and 22 nucleotides ofTNF-gamma. The restriction enzyme sites correspond to the restrictionenzyme sites in the bacterial expression vector pQE-9 (Qiagen). pQE-9was then digested with Bam HI and Xba I. The amplified sequences wereligated into pQE-9 and were inserted in frame with the sequence encodingfor the histidine tag and the RBS. The ligation mixture was then used totransform an E. coli strain available from Qiagen under the trademarkM15/rep 4 by the procedure described in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989).M15/rep4 contains multiple copies of the plasmid pREP4, which expressesthe lacI repressor and also confers kanamycin resistance (Kan^(r)).Transformants were identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies were selected. Plasmid DNA wasisolated and confirmed by restriction analysis. Clones containing thedesired constructs were grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture was used to inoculate a large culture at a ratio of 1:100 to1:250. The cells were grown to an optical density 600 (O.D.₆₀₀) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) wasthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells were grown an extra 3 to 4 hours. Cells were thenharvested by centrifugation. The cell pellet was solubilized in thechaotropic agent 6 M Guanidine HCl (Guanidine HCl concentrations ofgreater than or equal to 2.5 M were empirically found to result in ahigher level of purity of recovered recombinant protein). Afterclarification, solubilized TNF-gamma was purified from this solution bychromatography on a Nickel-Chelate column under conditions that allowfor tight binding by proteins containing the 6-His tag (Hochuli, E. etal., J. Chromatography 411:177-184 (1984)). TNF-gamma was furtherpurified by a second run on the Nickel-chelate column. TNF-gamma (90%pure) was eluted from the column in 6 M guanidine HCl pH 5.0 and for thepurpose of renaturation was dialyzed in PBS buffer. The expressionproduct was electrophoresed by SDS-PAGE, and the results may be seen inFIG. 5 where lanes labeled “M” contain molecular weight markers; lane 1is induced cell lysate; lane 2 is uninduced call lysate; lane 3 is theTNF-gamma protein after two Nickel-chelate column purifications; lane 4is the TNF-gamma protein after 1 column purification.

[0639] One of ordinary skill in the art will recognize that bacterialexpression vectors other than pQE-9 may also be used to expressTNF-gamma. One such preferred bacterial expression vector is pHE4-5.pHE4-5 may be obtained as pHE4-5/MPIFD23 plasmid DNA (this constructcontains an unrelated insert which encodes an unrelated ORF). ThepHE4-5/MPIFΔ23 plasmid was deposited with the American Type CultureCollection on Sep. 30, 1997 (Accession No. 209311). The ATCC is locatedat 10801 University Boulevard, Manassas, Va. 20110-2209, USA. Using theNde I and Asp 718 restriction sites flanking the unrelated MPIF ORFinsert, one of ordinary skill in the art could easily use currentmolecular biological techniques to replace the unrelated ORF in thepHE4-5/MPIFD23 plasmid with the TNF-gamma ORF, or variations thereof, ofthe present invention.

[0640] In a specific embodiment, a bacterial expression construct wasgenerated using the pHE-4 vector to express amino acid residues T-51through L-174 of SEQ ID NO:2.

[0641] In another specific embodiment, a bacterial expression constructwas generated using the pHE-4 vector to express amino acid residues T-58through L-174 of SEQ ID NO:2.

[0642] In a specific embodiment, a bacterial expression construct wasgenerated using the pHE-4 vector to express amino acid residues T-28through L-174 of SEQ ID NO:2.

[0643] In a specific embodiment, a bacterial expression construct wasgenerated using the pHE-4 vector to express amino acid residues T-30through L-174 of SEQ ID NO:2.

[0644] In a specific embodiment, a bacterial expression construct wasgenerated using the pQE-9 vector to express amino acid residues T-28through L-174 of SEQ ID NO:2 fused to a 5′ histidine tag.

[0645] In a specific embodiment, a bacterial expression construct wasgenerated using the pHE-4 vector to express amino acid residues L-72through L-172 of SEQ ID NO:20.

[0646] In a specific embodiment, a bacterial expression construct wasgenerated using the pHE-4 vector to express amino acid residues L-72through L-251 of SEQ ID NO:20 fused to a 5′ histidine tag.

[0647] In a specific embodiment, a bacterial expression construct wasgenerated using the pHE-4 vector to express amino acid residues L-72through L-251 of SEQ ID NO:20 fused to a 3′ histidine tag.

[0648] In a specific embodiment, a bacterial expression construct wasgenerated using the pHE-4 vector to express amino acid residues L-172through L-251 of SEQ ID NO:20 fused to a 5′ lacZ tag.

[0649] In a preferred embodiment, a polynucleotide encoding amino acidresidues Leu-72 through Leu-251 of a TNF-gamma-beta polypeptide (e.g.,as shown in SEQ ID NO:20 or as shown in SEQ ID NO:26) is cloned into abacterial expression vector (e.g., pHE-4, pHE4-0 or pHE4b-0) andexpressed in SG13009, W31 10 (ton A-) or M15/REP4 E. coli cells.

[0650] Also in a preferred embodiment, TNF-gamma-beta of the inventionis produced and isolated from SG13009, W3 110 or M15/REP4 E. colicultures using the following protocol.

Stage I. (SI)—Shake Flasks

[0651] Media contains Phytone, Yeast Extract, L-Methionine, and NaCl isprepared in shake flasks. The gene for aminoglycoside 3′phosphotransferase (kanR) is encoded on the expression plasmid sokanamycin is typically added to the seed medium to provide selectivepressure for cells maintaining the plasmid. MCB or WCB vials are thawedand used to inoculate shake flasks. The shake flasks are bottom-baffledand covered with a permeable top to maximize the transfer of gases(oxygen, carbon dioxide, etc.). The shake flasks are incubated in atemperature-controlled shaker/incubator. Growth in the flasks ismonitored using a spectrophotometer set in the visible wavelength. Oneor more 100, 150, 350, and/or 650 liter fermenters may be used for theproduction of TNF-gamma-beta. All product contact parts are constructedof Type 1 Borosilicate glass, 316 L stainless steel, medical gradeSilicone, Teflon or other FDA approved materials. When a sufficientoptical density (e.g., A₆₀₀=1-4) is attained in the seed vessel, theculture is used to inoculate either a production fermenter or a seedfermenter (SII). Typically, shake-flasks are used to inoculate smallproduction fermenters (<100 L). A seed fermenter (SII) is used toprepare the larger volume of inoculum required by larger productionfermenters.

Stage II (S2)—Seed Fermenter

[0652] Fermenters are engineered to provide a controlled environment forthe growth of bacteria. Many of the fermenter's functions arepreprogrammed and automated. They have agitators for mixing and have thecapability of controlling many conditions including temperature, pH anddissolved oxygen. All gasses enter and exit through a hydrophobic 0.2 umfilter to maintain sterility. Typically, the SII fermentation uses thesame medium as SI including kanamycin. Dissolved oxygen is controlledusing aeration, agitation, oxygen supplementation and back-pressure. pHis typically controlled using acid (e.g., phosphoric acid) and base(e.g., ammonium hydroxide) addition. Antifoam (e.g. Sigma Antifoam A) isused to neutralize foam. After inoculation with shake flasks, the SIIfermenter is grown until the desired optical density is reached (e.g.,A₆₀₀=1-4). The S2 fermenter is used to inoculate the productionfermenter.

Stage III (S3):—Production Fermenter

[0653] The production fermenter is batched with production medium (seetable 3 below) and heat sterilized. A defined, high cell densityfermentation medium is under development. After the fermenter hasequilibrated to process temperature, batch nutrients (see table 3 below)are added. Dissolved oxygen is controlled using aeration, agitation,oxygen supplementation and back-pressure. pH is typically controlledusing acid (e.g., phosphoric acid) and base (e.g., ammonium hydroxide)addition. S3 is inoculated by the culture from either a shake flasks ora seed fermenter. The cells are grown to a predetermined inductionoptical density (e.g., A₆₀₀=1-4). pHE4 plasmid is designed to suppressthe transcription of recombinant TNF-gamma-beta until desired. IPTG isadded to the fermentation to stop the suppression (induce) oftranscription of TNF-gamma-beta. At a specified time after induction,the fermentation is concluded. Time limits for S3 are under development.All operations involving open handling of cultures, medium, or productare conducted using aseptic techniques in laminar flow hoods. Liquidsare transferred in closed systems by overpressure using compressed airor a peristaltic pump to minimize the risk of introducing contaminants.TABLE 3 Fermentation Media and Supplements. Batch Medium currentlycontains: Batch Supplements currently contains: KH₂PO₄ Glucose Na₂HPO₄Zinc Sulfate 7-hydrate NaCl Ferric Chloride 6-hydrate NH₄Cl ManganeseChloride 4-hydrate Casamino Acids Cupric Sulfate 5-hydrate TryptoneCobalt Chloride 6-hydrate Yeast Extract Boric Acid L-CysteineHydrochloric Acid Tryptophan Magnesium Sulfate 7-hydrate L-HistidineMolybdic Acid Sodium Salt Dihydrate Uridine-HCl Monohydrate CaCl₂Thiamine-HCL

[0654] In specific embodiments, the concentrations of Batch Supplementsare varied. In one embodiment, the concentration of zinc sulfate7-hydrate is varied. In a specific embodiment, the concentration of zincsulfate is increased by 0.25-fold, 0.75-fold, 1-fold, 1.25-fold,1.5-fold, 1.75-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 7.5fold,10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 250-fold, 500-fold,750-fold, or 1000-fold.

[0655] TNF-gamma-beta is produced in the cytosol and maintained insidethe cell membrane. Cells are typically collected using centrifugation orfiltration. Cell paste is either processed immediately or is stored ator below −20° C. Stability studies of cell paste will be conducted toestablish expiration dating.

Recovery of TNF-gamma-beta Protein Step 1 Cell Harvest

[0656] The induced cell suspension is harvested between 4 and 8 hourspost IPTG induction. The TNF-gamma-beta containing cell paste isobtained with continuous flow centrifugation. Following centrifugation,the cell paste is used immediately or stored at −80° C.

Step 2 Cell Supernatant Production

[0657] The cell paste is suspended in 50 mM Tris-HCl buffer pH 8.0 in a10-fold volume of the cell paste. The cell suspension is homogenized andthe supernatant is produced by removal of cell debris with continuousflow centrifugation.

Purification of TNF-gamma-beta Protein Unless Stated Otherwise, theProcess is Conducted at 4-8° C. Step 1 Chromatography on QAE 550C Column

[0658] The supernatant is loaded onto a QAE 550C column (weak anionexchanger, TosoHaas) which is equilibrated with 50 mM Tris-HCl, pH 8.0containing 2 mM CaCl₂. The column is washed with the same buffer andthen the TNF-gamma-beta is eluted with 125 mM NaCl and 2 mM CaCl₂in 50mM Tris-HCl, pH 8.0. The elution is monitored by ultraviolet (UV)absorbance at 280 nm. Fractions are collected across the eluate peak,analyzed by SDS-PAGE, and appropriate fractions are pooled.

Step 2 Chromatography on Q- Sepharose Fast Flow (Q/FF)

[0659] The QAE pool is loaded onto a Q/FF (strong anion exchanger,Pharmacia) column equilibrated with 50 mM Tris-HCl containing 125 mMNaCl and 2 mM CaCl₂, pH 8.0. The column then is washed with the samebuffer. The TNF-gamma-beta is in the fraction of flow through. Theloading and wash are monitored by ultraviolet (UV) absorbance at 280 nm.

Step 3 Chromatography on Toyopearl Butyl 650S Column

[0660] The HQ50 pool is mixed with ammonium sulfate to produce a finalconcentration of 0.8 M and is loaded onto Toyopearl Butyl 650C(Hydrophobic interaction resin, TosoHaas) column equilibrated in 0.8 Mammonium sulfate in 100 mM Tris-HCl pH 7.3. The column is then washedwith a linear gradient elution of TNF-gamma-beta with 100 mM Tris-HCl pH7.3 followed by a 20% ethanol wash. The elution is monitored byultraviolet (UV) absorbance at 280 nm and conductivity. Fractions arecollected across the eluate peak, analyzed by SDS-PAGE. Appropriatefractions are pooled.

Step 4 Concentration on Toyopearl Butyl 650S

[0661] The Butyl purified TNF-gamma-beta is mixed with ammonium sulfateto produce a final concentration 0.8 M and is loaded onto a smallerToyopearl Butyl 650C (Hydrophobic interaction resin, TosoHaas) columnequilibrated in 0.8 M ammonium sulfate in 100 mM Tris-HCl pH 7.3.TNF-gamma-beta is eluted by stepwise with 100 mM Tris-HCl, pH 7.3.

Step 5 Chromatography on Superdex 200 Column

[0662] The Butyl concentrated TNF-gamma-beta is loaded onto a Superdex200 (Sizing Exclusive Chromatography, Pharmacia) column equilibrated in10 mM sodium citrate, 150 mM sodium chloride, pH 6.0. Fractions arecollected across the eluate peak and are analyzed by SDS-PAGE.Appropriate fractions (>90% purity) are pooled.

Step 6 Ultrafiltration, Filtration and Fill

[0663] The purified TNF-gamma-beta is placed into a 5 KD MW cutoffmembrane device to concentrate a target concentration. Then the proteinconcentration of purified TNF-gamma-beta is determined by absorbance at280 nm using TNF-gamma-beta extinction coefficient value (1 UV unit for1 mg/ml). TNF-gamma-beta formulation is adjusted to its final proteinconcentration with the appropriate buffer and filtered via 0.22micrometer filter under controlled conditions. The filtrate (bulksubstance) is stored in suitable sterilized container at 2-8° C.(short-term storage) or at or below −20° C. (long-term storage).

Example 2 Cloning and Expression of TNF-gamma Using the BaculovirusExpression System

[0664] The DNA sequence encoding the full length TNF-gamma protein, ATCCNo. 75927, was amplified using PCR oligonucleotide primers correspondingto the 5′ and 3′ sequences of the gene: The 5′ primer has the sequence5′-GCG CGG ATC CAC CAT GAG ACG CTT TTT AAG CAA AGT C-3′ (SEQ ID NO:15)and contains a Bam HI restriction enzyme site (in bold) followed by 24nucleotides of the TNF-gamma gene (the initiation codon for translation“ATG” is underlined). The 3′ primer has the sequence 5′-CGC GTC TAG ACTATA GTA AGA AGG CTC CAA AGA AGG-3′ (SEQ ID NO:16) and contains thecleavage site for the restriction endonuclease Xba I and 22 nucleotidescomplementary to the 3′ non-translated sequence of the TNF-gamma gene.The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with the endonucleases Barn mand Xba I and then purified again on a 1% agarose gel. This fragment wasdesignated F2.

[0665] The vector pA2 (modification of pVL941 vector, discussed below)was used for the expression of the TNF-gamma protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases Bam HI andXba I. The polyadenylation site of the simian virus SV40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E.coli was inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences were flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could have been used in place of pA2, such as pRG1,pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0666] The plasmid was digested with the restriction enzymes Bam HI andXba I and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA was then isolated from a 1% agarosegel using the commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA was designated V2.

[0667] Fragment F2 and the dephosphorylated plasmid V2 were ligated withT4 DNA ligase. E. coli XL1 blue cells were then transformed. Thesequence of the cloned fragment was confirmed by DNA sequencing.

[0668] 5 μg of the plasmid pBac TNF-gamma was cotransfected with 1.0 μgof a commercially available linearized baculovirus (“BaculoGoldbaculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0669] 1 μg of BaculoGold virus DNA and 5 μg of the plasmid pBacTNF-gamma were mixed in a sterile well of a microtiter plate containing50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium were added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added dropwise to the Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace' medium without serum. The plate was rocked back and forth tomix the newly added solution. The plate was then incubated for 5 hoursat 27° C. After 5 hours, the transfection solution was removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum was added. The plate was put back into an incubator andcultivation continued at 27° C. for four days.

[0670] After four days, the supernatant was collected and a plaque assayperformed essentially as described by Summers and Smith (supra). As amodification, an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0671] Four days after the serial dilution, the virus was added to thecells, blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

[0672] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-TNF-gamma at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of [35S]-methionine and 5 μCi [35S]-cysteine(Amersham) were added. The cells were further incubated for 16 hoursbefore they were harvested by centrifugation and the labeled proteinsvisualized by SDS-PAGE and autoradiography. FIG. 6 illustrates a gelwhere lanes 1 and 3 are the medium of the TNF-gamma and control culturesand lanes 2 and 4 are the cell lysates of the TNF-gamma and the controlcultures.

[0673] In a specific embodiment, a baculoviral expression construct wasgenerated using the pA2SPst vector to express amino acid residues V-25through L-174 of SEQ ID NO:2.

[0674] In a specific embodiment, a baculoviral expression construct wasgenerated using the pA2GP vector to express amino acid residues T-28through L-174 of SEQ ID NO:2 fused to a 5′ lacZ tag.

[0675] In a specific embodiment, a baculoviral expression construct wasgenerated using the pA2SPst vector to express amino acid residues A-61through L-251 of SEQ ID NO:20.

[0676] In a specific embodiment, a baculoviral expression construct wasgenerated using the pA2GP vector to express amino acid residues L-71through L-251 of SEQ ID NO:20.

[0677] In a specific embodiment, a baculoviral expression construct wasgenerated using the pA2GP vector to express amino acid residues L-71through L-251 of SEQ ID NO:20 fused to a 5′ lacZ tag.

[0678] In a specific embodiment, a baculoviral expression construct wasgenerated using the pA2 vector to express amino acid residues M-1through L-251 of SEQ ID NO:20.

Example 3 Expression of Recombinant TNF-gamma in COS Cells

[0679] The expression of plasmid, TNF-gamma-HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, an SV40 intron, and apolyadenylation site. A DNA fragment encoding the entire TNF-gammaprecursor and a hemagglutinin antigen (HA) tag fused in frame to its 3′end was cloned into the polylinker region of the vector. Therefore, therecombinant protein expression is under the direction of the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (I. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell37, 767). The fusion of HA tag to our target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

[0680] The plasmid construction strategy is described as follows: TheDNA sequence encoding TNF-gamma, ATCC #75927, was constructed by PCR onthe original EST cloned using two primers: the 5′ primer (SEQ ID NO:15)contains a Bam HI site followed by 24 nucleotides of TNF-gamma codingsequence starting from the initiation codon; the 3′ sequence 5′-CGC TCTAGA TCA AGC GTA GTC TGG GAC GTC GTA TGG ATA GTA AGA AGG CTC CAA AG-3′(SEQ ID NO:17) contains complementary sequences to Xba I site,translation stop codon, HA tag and the last 18 nucleotides of theTNF-gamma coding sequence (not including the stop codon). Therefore, thePCR product contained a Bam HI site, TNF-gamma coding sequence followedby HA tag fused in frame, a translation termination stop codon next tothe HA tag, and an Xba I site. The PCR amplified DNA fragment and thevector, pcDNAI/Amp, were digested with Bam HI and Xba I restrictionenzymes and ligated together. The ligation mixture was transformed intoE. coli strain SURE (available from Stratagene Cloning Systems, 11099North Torrey Pines Road, La Jolla, Calif. 92037) the transformed culturewas plated on ampicillin media plates and resistant colonies wereselected. Plasmid DNA was isolated from transformants and examined byrestriction analysis for the presence of the correct fragment. Forexpression of the recombinant TNF-gamma, COS cells were transfected withthe expression vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch,T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989)). The expression of the TNF-gamma HA proteinwas detected by radiolabelling and immunoprecipitation method. (E.Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, (1988)). Cells were labeled for 8 hours with[35S]-S-cysteine two days post transfection. Culture media were thencollected and cells were lysed with detergent (RIPA buffer (150 mM NaCl,1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5; Wilson, I.et al., Id. 37:767 (1984)). Both cell lysate and culture media wereprecipitated with an HA-specific monoclonal antibody. Precipitatedproteins were then analyzed on 15% SDS-PAGE gels.

[0681] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4 vector to express amino acid residues M-1through L-251 of SEQ ID NO:20.

[0682] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4SPst vector to express amino acid residues A-61through L-251 of SEQ ID NO:20.

[0683] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4 vector to express amino acid residues L-72through L-251 of SEQ ID NO:20 fused to the Fc region of humanimmunoglobulin, as described supra.

[0684] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4SP vector to express amino acid residues L-72through L-251 of SEQ ID NO:20 fused to lacZ at the amino terminus.

[0685] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4 vector to express amino acid residues M-1through L-174 of SEQ ID NO:2.

[0686] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4SP vector to express amino acid residues T-28through L-174 of SEQ ID NO:2.

[0687] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4SPst vector to express amino acid residues V-25through L-174 of SEQ ID NO:2.

[0688] In a specific embodiment, a mammalian expression construct wasgenerated using the pC4SP vector to express amino acid residues T-28through L-174 of SEQ ID NO:2 fused to lacZ at the amino terminus.

Example 4 Expression Pattern of TNF-gamma in Human Tissue

[0689] RNA blot analysis was carried out to examine the levels ofexpression of TNF-gamma in human tissues. Total cellular RNA sampleswere isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023South Loop East, Houston, Tex. 77033). About 2 μg (for the RNA blot ofFIG. 3A) of total RNA isolated from each human tissue specified wasseparated on 1% agarose-formaldehyde gel and blotted onto a nylon filter(Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring HarborPress, (1989)). The labeling reaction was done according to theStratagene Prime-It kit with 50 ng TNF-gamma cDNA, to produce[32P]-labeled TNF-gamma cDNA. The labeled DNA was purified with aSelect-G-50 column (5 Prime-3 Prime, Inc. 5603 Arapahoe Road, Boulder,Colo. 80303). The filter was then hybridized with radioactive labeledfull-length TNF-gamma gene at 1,000,000 cpm/ml in 0.5 M NaPO4, pH 7.4and 7% SDS overnight at 65° C. After being washed twice at roomtemperature and twice at 60° C. with 0.5×SSC, 0.1% SDS, the X-ray filmwas then exposed to the blot at −70° C. overnight with an intensifyingscreen. The message RNA for TNF-gamma is abundant in kidney.

[0690] The same reaction was done to obtain the results shown in FIG.3B, with the exception that 10 μg poly-A RNA isolated from the indicatedtissues was used. In this experiment, the messenger RNA encodingTNF-gamma is expressed predominantly in HUVEC cells (FIG. 3B; lane 9),but not in other cell lines examined; for example; lane 1 is CAMA1(breast cancer); lane 2 is AN3CA (uterine cancer); lane 3 is SK.UT.1(uterine cancer); lane 4 is MG63 (osteoblastoma); lane 5 is HOS(osteoblastoma); lane 6 is MCF7 (breast cancer); lane 7 is OVCAR-3(ovarian cancer); lane 8 is CAOV-3 (ovarian cancer); lane 10 is AOSMIC(smooth muscle); and lane 11 is foreskin fibroblast.

[0691] Northern blot analyses were also performed to determine therelative expression level of the TNF-gamma RNA with respect to theproliferation state of HUVEC cell cultures. In these experiments,identical amounts of total RNA isolated from HUVEC cells (15 μg) wereelectrophoretically separated and blotted as described above. RNA wasisolated from cultures 1, 2, 3, 4, 6, and 7 days post-seeding. Asillustrated in FIG. 4, TNF-gamma RNA (labeled “VEGI”) was only seen atlow levels in newly seeded cultures (days 1, 2, and 3). However,expression of TNF-gamma RNA was apparent as the HUVEC cells in thecultures began to reach confluence (days 4, 6, and 7). These experimentsindicate that TNF-gamma expression increases as cells in a culture ortissue begin to reach the quiescent state of non- or reduced-proliferation.

[0692] In other experiments performed essentially as described above,the TNF-gamma-alpha transcript has been detected in many different humantissues, e.g., placenta, lung, kidney, skeletal muscle, pancreas,spleen, prostate, small intestine, and colon. Further experiments haveshown that expression of the TNF-gamma-alpha molecule was greatest in asubset of endothelial cells, such as human umbilical vein endothelialcells (HUVECs) and human uterine myometrial microvascular endothelialcells (HMMVECs), but not in human pulmonary artery endothelial cells(HPAEC), human iliac artery endothelial cells (HIAEC), or human coronaryartery endothelial cells (HCAEC). The transcript for TNF-gamma-beta hasalso been detected in placenta, lung, kidney, prostate, small intestine,stomach, liver, kidney, and pancreas, HUVECs, HMMVECs, human aorticendothelial cells (HAECs), and human microvascular endothelial cells(HUMECs).

Example 5 Ability of Recombinant TNF-gamma to Inhibit WEHI 164, ABAE,and L929 Cell Growth, and to Induce Cell Adhesion in HL-60 Cells

[0693] The adherent target cells were prepared from confluent culturesby trypsinization in PBS, and non-adherent target cells were harvestedfrom stationary cultures and washed once with medium. Target cells weresuspended at 3×105 cells/ml in medium containing 10% FCS. 0.1 mlaliquots were dispensed into 96-well flat-bottomed microtiter platescontaining 0.1 ml serially diluted test samples of cells (WEHI 164 andL929). Incubation was continued for 70 hours. TNF-alpha, TNF-beta andbacterially-produced TNF-gamma were added at a 0.5 μg/ml concentration.The cytotoxicity and proliferation activity was quantified using an MTSassay performed by the addition of 20 μl of MTS and phenazinemethosulfate (PMS) solution to each well. After a three hour incubation,the OD at 492 nm was measured by an ELISA plate reader. The OD492 isproportional to the number of viable cells in the wells. The percent ofcytotoxicity was calculated as follows: %cytotoxicity=(100−ODexperimental/ODcontrol)×100. The photographs weretaken after 72 hours. As shown by FIGS. 7A and 8, TNF-gamma induced amorphology change which appeared as dark round cells (indicating killedcells).

[0694] In the graph of FIG. 7B, the assay was performed as describedabove, however, increasing amounts of TNF-alpha, TNF-beta and TNF-gammawere added to the cultures. The results indicate that TNF-gamma is adose-dependent inhibitor of the growth of the endothelial cell line WEHI164, but not of the fibroblast cell line L929 (FIGS. 8 and 9).

[0695] A truncated form of the TNF-gamma polypeptide consisting of aminoacids 12-147 of the complete TNF-gamma amino acid sequence shown as SEQID NO:2 (designated TNF-gamma12-147) was also used to examine the effectof TNF-gamma on endothelial cell growth. Treatment of adult bovineaortic endothelial (ABAE) cells with TNF-gamma12-147 resulted in nearlycomplete inhibition of the growth of cells in the ABAE culture, but notof cells in the breast cancer cell lines MDA-MB-435 or MDA-MB-231 (FIG.10; TNF-gamma is designated “VEGI” in this figure). Nearly completeinhibition of the growth of the endothelial cells was achieved at 10μg/ml TNF-gamma39-174, with a half-maximum inhibitory concentrationvalue (IC50) of approximately 1 μg/ml (approximately 70 nM).

[0696] To test adhesion ability of TNF-gamma, HL-60 cells were used andcell adhesion and cell-cell contact were measured by observation underthe microscope and scored subjectively by two independent investigators.FIG. 11 illustrates the ability of TNF-gamma to induce cell adhesion.Cultures which were not treated with TNF-gamma contained cells which hadspread throughout the culture dish. However, cultures which were treatedwith TNF-gamma, contained cells which were clearly aggregated together.

Example 6 Measurement of Apoptosis Ability of TNF-gamma

[0697] In a first incubation step, anti-histone antibody was fixedadsorptively on the wall of a microtiter plate module. Subsequently,non-specific binding sites on the wall were saturated by treatment withincubation buffer (e.g., blocking solution). During the secondincubation step, the nucleosomes contained in the WEHI 164 cell sampletreated with the TNF-alpha, TNF-beta or bacterially-produced TNF-gammawere bound via their histone components to the immobilized anti-histoneantibody. In the third incubation step, anti-DNA-peroxidase (POD)reacted with the DNA component of the nucleosomes. After removal of allunbound peroxidase conjugate by a washing step, the amount of peroxidaseretained in the immunocomplex was determined spectrophotometricallyusing the substrate ABTS (2,2′-azino-di-[3-ethylbenzthiazolinesulfonate]). Anti-histone antibody reacted with the histones H1, H2A,H2B, H3, and H4 from the sample. Anti-DNA POD antibody bound to single-and double-stranded DNA. Therefore, the ELISA allowed the detection ofmono- and oligonucleosomes and may be applied to measure apoptotic celldeath. The level of cell death was measured by the amount of cytoplasmichistone-associated DNA fragments which was indicated by the ratio of theabsorbances observed at 405 and 490 nm (A405/A490). The results of theseexperiments are illustrated in FIG. 12 (See Boehringer MannheimCatalogue, 0990 C 93 2 1541170).

[0698] As shown in FIG. 12, WEHI 164 cells were induced to undergoincreasingly high levels of apoptosis, resulting in cell death, in thepresence of increasing amounts of TNF-gamma. This effect was alsoobserved in the presence of increasing amounts of the control TNF-betaor in the presence of any of the analyzed levels of the controlTNF-alpha.

Example 7 Receptor Binding Assay Using TNF-gamma

[0699] TNF-alpha and bacterially-produced TNF-gamma were purified byNi-NTA affinity chromatography using the 6-His tag fused to the terminusof the recombinant proteins. 1 μg/well of either protein was added to anickel chelate-coated 96-well plate (Xenopore Corp.) and incubated for 2hours. After washing three times, 100 ng of human soluble TNF receptors(specifically, sTNF RI or sTNF RH) was added to each well and incubatedfor 2 hours. The plate was then washed three times and alkalinephosphatase-labeled polyclonal antibodies raised against either sTNF RIor sTNF RII was added in a total volume of 200 μl. An aliquot ofsubstrate solution (200 μl) was then added to each well and the platewas incubated for an additional 2 hours. The OD was then measured usingan ELISA reader (at a test wavelength of 450 nm and a correctionwavelength of 590 nm). The results shown in FIG. 13 illustrate thatTNF-gamma does not bind significantly to sTNF-receptors when compared tothe control binding observed with TNF-alpha.

Example 8 Expression via Gene Therapy

[0700] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask. At this time, fresh media is added (e.g., Ham's F12 media,supplemented with 10% FBS, penicillin, and streptomycin). The culture isthen incubated at 37° C. for approximately one week. At this time, freshmedia is added and subsequently changed every 2-3 days. After anadditional two weeks in culture, a monolayer of fibroblasts will haveemerged. The monolayer is trypsinized and scaled into larger flasks.

[0701] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988)), which isflanked by the long terminal repeats of the Moloney murine sarcomavirus, is digested with Eco RI and Hind III, and, subsequently, treatedwith calf intestinal phosphatase. The linear vector is fractionated onagarose gel and purified using glass beads.

[0702] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an Eco RI site and the3′ primer includes a Hind III site. Equal quantities of the Moloneymurine sarcoma virus linear backbone and the amplified Eco RI and HindIII fragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0703] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0704] Fresh media is added to the transduced producer cells, and,subsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells. This media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it may be necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0705] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

Example 9 In Vitro Angiogenesis Assay

[0706] This assay was used to determine the relative ability ofTNF-gamma12-147 to inhibit the FGF-2-induced formation of capillary-liketubular structures in cultures of adult bovine aortic endothelial (ABAE)cells. Three-dimensional collagen gel plates (24-well) were prepared byaddition of 0.5 ml chilled solution of 0.7 mg/ml of rat tail type Icollagen (Becton Dickinson Labwares, Bedford, Mass.) to each wellcontaining 1×DMEM and adjusting to neutral pH with NaHCO3. Afterformation of collagen gel (about 1-2 mm thickness), ABAE cells wereseeded at 5×104 cells/well. The cultures were maintained in a humidified5% CO₂ incubator at 37° C. in DMEM containing 10% calf serum (HyClone,Logan, Utah) supplemented with L-glutamine (2 mM) until the culturesreached confluence. The medium was then replaced with fresh mediumcontaining 20 ng/ml of FGF-2. The effect of TNF-gamma12-147 as aninhibitor of FGF-2-induced formation of capillary-like tubularstructures in ABAE cultures was analyzed by supplementing the culturemedium with 0.1, 0.3, 1, 3, or 10 μg/ml of TNF-gamma12-147. All cultureswere then maintained at 37° C. for an additional 48 hours and thendiscontinued by fixation with cold methanol (−20° C.).

[0707] The abundance of capillary-like structures formed by ABAE cellswas analyzed by using a Kotron IBAS Image Analyzer assisted with aHamamatsu C2400 video camera and a Zeiss Axioshop microscope. Theabundance of the capillary-like structures were measured as percentagesof the white areas over the total areas measured. As a control, the EC50value for the angiogenic factor FGF-2 to stimulate in vitro angiogenesiswas about 5 ng/ml. As a further control, a maximum stimulatory effectwas observed at 10 ng/ml of FGF-2.

[0708] As shown in FIG. 14 (in which TNF-gamma is designated “VEGI”),observable inhibition of FGF-2-induced tube formation in ABAE cultureswas observed by the addition of 1, 3, and 10 μg/ml of TNF-gamma12-147(labeled as VEGI). The IC50 value for the inhibition of FGF-2-inducedtube formation was approximately 1 μg/ml, which was similar to thatobserved for the inhibition of endothelial cell growth (see Example 5).

Example 10 Chicken Embryonic Chorioallantoic Membrane (CAM) AngiogenesisAssay

[0709] The CAM assay was carried out essentially as described by Nguyenand colleagues (Microvasc. Res. 47:31-40 (1994)) and Iruela-Arispe andDvorak (Thromb. Haemost. 78:672-677 (1997)). The method is based on thegrowth of new capillary vessels into a collagen gel pellet placeddirectly on the chorioallantoic membrane (CAM). Angiogenic factors suchas endostatin (2 micrograms), FGF-2 (100 ng), VEGF (250 ng), or bFGF(10, 500, and 1000 ng) were embedded in collagen gel pellets and placedin contact with the CAM. Quantification of angiogenesis in the gels wascarried out 24 hours after the placement of the gel pellets by using aNikon fluorescence microscope. The images were transferred to a Power PC100 AV, using a CCD Sony camera. Fluorescence intensity was evaluatedwith NH Image 1.61 software. Fluorescence intensity for the positivecontrols (which contained an angiogenic factor alone) was considered asthe maximum angiogenic response, and set, arbitrarily, at 100. Due tothe variability of the assay, inhibition greater than 20% was consideredsignificant.

[0710] As an experimental determination of the effect of TNF-gamma onthe FGF-2- or VEGF-induced angiogenesis, bacterially-produced TNF-gamma(250 ng) was mixed with either FGF-2 (100 ng) or VEGF (250 ng) andembedded in collagen gel pellets. The pellets were then placed incontact with the CAM as described above. As shown in FIG. 15 (in whichTNF-gamma is designated “VEGI”), TNF-gamma markedly inhibited newcapillary growth into collagen gels.

[0711] In another experiment, 50, 100, 250, 500, 1000 or 2000 ng ofTNF-gamma-beta were analyzed for a reduction in bFGF-induced stimulationof neovascularization in the CAM assay. By the 72 hour timepoint in thisexperiment, 1000 and 2000 ng of TNF-gamma-beta reduced bFGF-stimulatedangiogenesis to a level indistinguishable from control levels notreceiving bFGF.

Example 11 In Vivo Tumorigenicity Assay

[0712] An in vivo analysis of the potential effect of TNF-gamma onangiogenesis was performed using a xenograft tumor model. In thisexperimental approach, one million human breast carcinoma cells(MDA-MB-231 or MDA-MB-435) were injected into the mammary fat pad offemale nude mice either alone or mixed with chinese hamster ovary (CHO)cells transfected with TNF-gamma or CHO cells transfected only with theCHO-vector (5×10⁶ cells per mouse). The TNF-gamma polypeptide expressedin these experiments consisted of the polypeptide shown as SEQ ID NO:2excluding the N-terminal 22 amino acids. The N-terminal 22 amino acidsof this TNF-gamma mutein were replaced by the secretory signal peptideof human interleukin-6 (Hirano, T., et al., Nature 324:73-76 (1986)).

[0713] Mice which were coinjected with human breast carcinoma cells andeither TNF-gamma-expressing CHO cells or vector-transfected CHO cellswere then randomized and tumors were measured twice weekly. The tumorsize was assessed by measuring perpendicular diameters with a caliperand calculated by multiplying the measurements of diameters in twodimensions. Data are presented in FIGS. 16A and 16B as themean+/−standard deviation of six mice in each group.

[0714] Results presented in FIG. 16A and 16B (in which TNF-gamma isdesignated “VEGI”) illustrate the sizes of the MDA-MB-231 andMDA-MB-435, respectively, xenograft tumors (mm²) as a function of time(days postinoculation). Tumors were measured beginning on day zero andapproximately at 5 day intervals through approximately the twenty-eighthday. In each case, tumors which resulted from breast carcinoma cellscoinjected with TNF-gamma-expressing CHO cells (represented by theclosed circles in FIGS. 16A and 16B) remained significantly smaller insize than those which resulted from breast carcinoma cells coinjectedwith vector-only CHO cells (represented by the open circles in FIGS. 16Aand 16B).

Example 12 Induction of NF-kappaB and c-Jun Kinase (JNK) by TNF-gamma

[0715] Activation of cellular NF-kappaB is preceded by thephosphorylation, ubiquitination, and ultimate degradation of anendogenous NF-kappaB inhibitor molecule designated IkBa. Degradation ofthe inhibitor allows the p65 subunit of NF-kappaB to translocate to thenucleus where it can act as a transcriptional regulator. For thisreason, a electrophoretic mobility shift analysis (EMSA) is anappropriate method for analyzing activation of cellular NF-kappaB bytreatment of cultured cells with TNF-gamma.

[0716] In these analyses cells (2×10⁶ per ml) were treated withdifferent concentrations (0.1-1.0 μg/ml) of bacterially-producedTNF-gamma at 37° C. for 12 hours. Nuclear extracts were then preparedfrom the cultured cells and EMSA was performed as is well-known in theart and essentially as described (Singh, S. and Aggarwal, B. B. J. Biol.Chem. 270:10631-10636 (1995)).

[0717] Treating U-937 cells with TNF-gamma for 12 hours resulted inincrease in DNA-binding by the p65 subunit of NF-kappaB. Peak activationof DNA-binding by p65 was observed when U-937 cells were treated with 1μg/ml TNF-gamma for 12 hours. However, treatment of U-937 cells with aslittle as 0.2 μg/ml TNF-gamma for 12 hours resulted in an observableincrease in p65 DNA-binding. TNF-gamma was observed to activate p65DNA-binding over basal levels from 30 minutes to 18 hours after theinitiation of treatment in U-937 cells.

[0718] These experiments were elaborated by determining a degradationprofile for 1-kappaBa in U-937 cells in response to treatment withTNF-gamma. A time course of 1-kappaBa degradation was determined byWestern blot analysis, a technique that is well-known by one of ordinaryskill in the art and has been described by Singh and Aggarwal (J. Biol.Chem. 270:24995-25000 (1995)). 1-kappaBa was completely degraded whenU-937 cells were treated with 0.1-1.0 μg/ml TNF-gamma for 12 hours.

[0719] The cellular kinase designated c-Jun kinase (JNK) is an earlyevent in cellular activation. The activation of JNK by TNF-gamma wasanalyzed as an additional method of determining cellular reaction totreatment with TNF-gamma. The JNK kinase activation assay is well-knownby one of skill in the art and has been described by Derijard andcolleagues (Cell 76:1025-1029 (1994)). After treatment of U-937 cellswith 0.1 to 3.0 μg/ml of TNF-gamma for 12 hours, the cells wereharvested and assayed for JNK kinase activity. By 6 and 12 hours, JNKactivity had increased 2- and 3.6-fold, respectively.

Example 13 Effect of TNF-gamma in Treating Adjuvant-induced Arthritis inRats

[0720] An analysis of the use of TNF-gamma to treat rheumatoid arthritis(RA) may be performed through the use of an adjuvant-induced arthritis(AIA) model in rats. AIA is a well-characterized and reproducible animalmodel of rheumatoid arthritis which is well-known to one of ordinaryskill in the art (Pearson, Ann. Rheum. Dis. 15:379 (1956); Pearson &Wood, Arthritis Rheum. 2:440 (1959)). TNF-gamma is expected to inhibitthe increase in angiogensis or the increase in endothelial cellproliferation required to sustain the invading pannus in bone andcartilage observed in this animal model of RA. Lewis and BB rats(available from Charles River Lab, Raleigh, N.C. and the University ofMassachusetts Medical Center, Worcester, Mass.) are used as the commonand responsive strains for adjuvant-induced arthritis in theseexperiments.

[0721] Initiation of the arthritic condition is induced by theintradermal injection of 0.1 ml adjuvant (5 mg/ml) into the base of thetail. Groups of 5 to 6 rats receive either 0.1 to 1.0 mg/kg TNF-gamma orvehicle intra-articularly 20 days after the injection of adjuvant. Atthis timepoint, acute inflammation reaches a maximal level and chronicpannus formation will have just begun. The effect of TNF-gamma on pannusformation is analyzed radiologically once each week after day 15following adjuvant challenge essentially as described by Taurog andcolleagues (J. Exp. Med. 162:962 (1985)). Briefly, rats are anesthetizedwith ether or chloral hydrate and positioned so that both hind limbs areX-rayed together. The X-ray films is examined blindly using a scoringsystem of 0-3 for periosteal reaction, bony erosions, joint spacenarrowing and destruction. When there is a significant amount of jointdamage in vehicle-treated rats, the animals are sacrificed. At thispoint, the paws are evaluated histologically for the relative degree oftissue damage and for the therapeutic effect TNF-gamma has elicited onthese joints.

[0722] Finally, TNF-gamma- and vehicle-treated animals undergo aclinical evaluation twice per week to assess hind paw volume using aplethysmometer system and body weight.

Example 14 DR3 Ligand (TNF-gamma) is a Novel Anti-tumor CytokineExisting in Two Different Forms and Differentially Expressed inDifferent Tissues and Cells Background

[0723] TNF (tumor necrosis factor) superfamily members play veryimportant roles in cell activation, proliferation, differentiation,apoptosis, cytotoxicity and immune regulation. Members of TNF ligand andreceptor superfamily are often overexpressed in various human cancercells and/or activated lymphocytes, their extracellular accessibilitymakes them excellent potential targets for specific antitumor therapyand immunomodulating therapy. Over the past few years the list ofmolecules belonging to the TNF receptor and ligand superfamily has grownrapidly. The TNF ligand family of cytokines consist of over 13 type IItransmembrane proteins (except TNF-beta), the TNF receptor superfamilyconsist of over 18 type I transmembrane proteins except OPG, also knownas OCIF or TR1, which is a secreted protein, and TRID/DcR1I/TRIAL-R3,which is a GPI-linked cell surface molecule.

[0724] everal TNF receptor superfamily members as well as some of theintracellular signal transducers involved in apoptosis contain a stretchof amino acids, approximately 60 to 80 amino acid long, referred to asthe “death domain”. These death domain-containing receptors, such asTNFR1, Fas/Apo-1/CD95, DR3 (also known as Wsl, Apo3, TRAMP or LARD),DR4, DR5 or TRAIL-R2, upon activation by their ligands, recruit variousproteins that mediate cell death through the death domain. Theseproteins in turn recruit other proteins via their death domains or deatheffector domains to transduce the death signal. TNFR1 is expressed inmost tissues and cell types and is involved in transducing three majortypes of signals: activation of the transcription factor NF-kappaB,c-jun N-terminal protein kinase and apoptosis. Whereas Fas is expressedin lymphocytes, liver, heart, lung, kidney, and ovary. In contrast, DR3is predominantly expressed in spleen, thymus, and peripheral bloodlymphocytes. The ligand for DR3 has not yet been identified. DR3interacts with TRADD, associates with RIP ordinarily only weakly, butassociates strongly when TRADD is overexpressed. In the presence ofTRADD, it also associates strongly with FADD. These results suggest thatthe mechanism of DR3-induced apoptosis is similar to that induced by Fasand TNFR1. Like TNFR1, DR3 also activates NF-kappaB.

[0725] We have identified several novel TNF receptor and ligandsuperfamily members using several search strategies. One novel TNF-likeligand, TNF-gamma, was predominantly expressed in endothelial cells.Although TNF-gamma shares some of the activities TNF, it does not bindto TNFR1 and TNFR2, indicating that TNF-gamma binds to a distinctreceptor. Here we show that TNF-gamma binds to DR3 in severalreceptor-ligand binding assays. Interestingly, TNF-gamma exists in twodifferent forms which are differentially expresses in different cellsand tissues.

Results and Discussion

[0726] We have identified several novel TNF receptor and ligandsuperfamily members from HGS database which contains over 1.5 millionESTs from over 620 cDNA libraries. One novel TNF-like ligandpredominantly expressed in an endothelial cell library exhibited 20-30%sequence homology to other members of the TNF family. The protein wasnamed TNF-gamma-alpha (or VEGIa for Vascular Endothelial derived tumorGrowth Inhibitor alpha). Subsequent database analysis and libraryscreening identified a novel splicing variant of TNF-gamma-alpha,designated TNF-gamma-beta (or VEGIbeta). This isoform was foundpredominantly in cDNA libraries of TNFalpha- and IL-1-inducedendothelial cells, monocyte and activated T-cells. The cDNA forTNF-gamma-alpha encodes 174 amino acid residues and TNF-gamma-betaencodes 251 amino acids. Both proteins have characteristics of type IItransmembrane proteins. They only differ at the N-terminus whichcorresponds to the intracellular and transmembrane domains (FIG. 18A-Dand 19).

[0727] Recombinant TNF-gamma induces apoptosis in several cell linessuch as bovine pulmonary artery endothelial cells and adult bovineaortic endothelial cells. [Bovine pulmonary artery endothelial cellswere incubated with various concentrations of TNF-gamma for 48 hours.The apoptosis was assessed by nuclear staining with Hoechst 33342fluorescence dye (10 mg/ml).] TNF-gamma also induces nuclearfactor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) activation,inhibits angiogenesis in vitro. [U937 cells were transfected usinglipofectamine (following manufacturer's instruction) with 0.2 mg ofreporter plasmid (NF-kappaB-SEAP). The transfected U937 cells werecollected and added to the 96-well plate (200 ml/well) with variousconcentrated of TNF-gamma. After Incubation at 37° C. for 72 hr, theNF-kappaB activity was measured with luminometer at absorbance of 450nm.]

[0728] To identify the novel receptor and ligand pairs, severalreceptor-ligand binding assays were established. Recombinant solubleTNF-gamma containing the entire ectodomain binds to DR3-Fc fusionprotein immobilized on BIAcore chip, purified DR3-Fc also binds toBIAcore chip immobilized with TNF-gamma. [Purified DR3-Fc or TNF-gammawas analyzed on a BIAcore instrument flowcell derivatized with TNF-gammaor DR3-Fc. The shown data represents the net bound (off-rate) region ofthe plot after binding of TNF-gamma to immobilized DR3-Fc receptor, orbinding of DR3-Fc to immobilized TNF-gamma, which is measured inrelative mass units (RU) versus time. The binding conditions wereperformed at high receptor chip densities under diffusion-limitedconditions.] Using immunoprecipitation techniques, recombinant TNF-gammawas co-immunoprecipitated by DR3-Fc, but not LTbR-Fc immunoadhesins.[The Fc-extracellular domains of DR3 or Fc alone and the correspondingligands were prepared and binding assays were performed as describedelsewhere. The respective Fc-fusions were precipitated with proteinG-Sepharose and co-precipitated soluble ligands were detected byimmunoblotting with anti-TNF-gamma antibody. Blotting and detection wasperformed as described in BM Chemiluminescence Western Blotting kitprotocol.]

[0729] o further demonstrate the interaction between DR3 and TNF-gamma,we screened several cell lines for cell surface expression of TNF-gammausing polyclonal antibody to recombinant soluble TNF-gamma. Consistentwith the Northern blot analysis, peripheral blood mononuclear cells(PBMC) and human umbilical vein endothelial cells (HUVEC) expressTNF-gamma on the cell surface by immunostaining with antibody toTNF-gamma. [Cells were collected by trypsinization or aspiration, andcentrifuged at 1500-2000 rpm for 5 min. The cell pellets wereresuspended and washed in 5 ml ice-cold PBS twice. The cells wereincubated for 30 min at 40° C. with antibody (10 mg/ml ) to TNF-gamma todetected expression of TNF-gamma on cell surface, with DR3-Fc or LTbR-Fc(10 mg/ml) for receptor and ligand binding in the binding buffer (HBSScontaining 10% BSA, 20 mM HEPES, pH 7.2, 0.02% NaN3). Purified human IgG(25 mg/ml) was used as control. Cells were then washed and stained withphycoerythrin (PE) conjugated to goat anti-rabbit or anti-human IgG at20 mg/ml. Fluorescence was analyzed by a FACscan flow cytometer (BectonDickinson, Mountain View, Calif.).] Two tumor cell lines(MC-38/TNF-gamma and MDA-23 1/TNF-gamma) transfected with TNF-gamma alsoexpress TNF-gamma on the cell surface. FACS analysis showed that here isa shift in the most population following exposure MC-38/TNF-gamma cellsto DR3-Fc, indicating cell-surface binding between TNF-gamma and DR3.Similarly, a shift in the MDA-231 cells transfected with TNF-gamma wasobserved. In addition, DR3-Fc protein also binds to HUVEC cells andPBMC. It is noteworthy that DR3 expression and TNF-gamma binding to PBMCdeclined after prolonged stimulation with PHA. As predicated, DR3-Fcinhibits the TNF-gamma induced NF-KB activated in a dose-dependentmanner. [U937 cells were transfected using lipofectamine (followingmanufacturer's instructions) with 0.2 mg of reporter plasmid(NF-kB-SEAP). The transfected U937 cells were collected and added to the96-well plate (200 ml/well) with various concentration of DR3-Fcreceptor and 100 ng/ml of TNF-gamma. After incubation at 37° C. for 72hr, the NF-KB activity was measured with luminometer at absorbance of450 nm.]

[0730] TNF-gamma maps to the chromosomal location within band 9q32. Thischromosomal location is close to CD30L (9q33), but is different from thegenes for TNFalpha, LTalpha and LTbeta which are tightly linked withinthe MHC complex on chromosome 6. Interestingly, the TNF-gamma receptor,DR3, was assigned to the long arm of chromosome 1, region p36.2, islocalized to a region where CD30, TNFR2 and OX40 have been mapped.

[0731] Consistent with the role of TNF-gamma and DR3 in apoptosis andimmune regulation as well as interaction of DR3 with TNF-gamma, localproduction of TNF-gamma caused complete tumor suppression in vivo in asyngeneic MC-38 murine colon cancer models. In the same animal model,local production of soluble DR3, which may block TNF-gamma function,promotes tumor growth. [The full-length TNF-gamma and extracellulardomain of DR3 was cloned into pcDNA3 expression vector and transfectedto MCA 38 cells, respectively. After selection and cloning, three clonesfrom each constructs were picked for tumorigenicity study. MCA 38 cells(1×10⁶ cells/mouse) expressing TNF-gamma or DR3 extracellular domainwere injected into C57BL6/6 mice. The tumor size was assessed bymeasuring perpendicular diameters with a caliper and calculated bymultiplying the measurements of diameters in two dimensions. Data arerepresented as the mean+/−SD of 6 mice in each group.] It is clear thatmost immune cells and cancer cells can express more than one TNFreceptor (even more than one death receptor) and ligand superfamilymember. The existence of multiple receptors for one ligand or multipleligands for one receptor, and multiple splicing variant forms ofreceptor or ligand suggests an unexpected complexity in the regulationof apoptosis and immune function. These receptors and ligands appear tobe functionally redundant, but their expression patterns are different,suggesting a distinct tissue or cell specific involvement in aparticular function. Moreover, the expression of these ligands andreceptors may differ at the level of individual cell types withintissues and the expression level on the same cell type may also differ.

[0732] It is estimated that 10% of genes can be alternatively spliced,but in many cases the function of proteins produced remains obscure. Toexamine the potential functional significance of the two splicingvariants of TNF-gamma, PCR analysis was performed in over 100 cDNAlibraries. These results are shown in the following table:

[0733] Differential expression pattern of DR3, TNF-gamma-alpha, andTNF-gamma-beta Library DR3 TNF-γα TNF-γβ Library DR3 TNF-γα TNF-γβNormal Tissue Abnormal tissue and cell Liver + + Hepatocellular tumor +Lymph node + + Hodgkin's lymphoma + Tonsil + Rhabdomyosarcoma + Bonemarrow + Nasal polyps Spleen + Spleen, metastatic melanoma Heart +Spleen, chronic Thymus + + lymphocytic leukemia Pericardium + Healingwound (skin) + + Brain + B-cell lymphoma Lung + HemangiopericytomaSkeletal muscle Pancreas tumor + Placenta + Burned skin + Prostate +Prostate cancer, stage C Pituitary U937 cell + Testis + + Ovariantumor + Colon Colon cancer, metasticized + + Pancreas + to liverKidney + Colon Cancer Kidney cortex + Crohn's disease Pulmonary Rejectedkidney + + Adipose + + T-cell lymphoma + Ovary + + Ovary tumorCerebellum Endometrial tumor Hippocampus Skin tumor HyperthalamusPancreatic carcinoma + Olfactory epithelium + + Jurkat cells + Striatumdepression + Hela cell line + + Pineal gland LNCAP + 0.3 nM androgen +LNCAP + 30 nM androgen + + Library DR3 TNF-ga TNF-gb Library DR3 TNF-gaTNF-gb Fetal tissue Normal cell 8 week embryo + + HUVEC. + + + 9 weekembryo + Dermal endothelial, + Fetal brain + + + Resting T cell Fetalkidney + + Activated T cell (12 hr) Fetal heart + + + Activated T cell(16 hr) + Fetal thymus + Activated T cell (24 hr) + + Fetal lung + + Tcell helper I Fetal liver + T cell helper II + Fetal spleen + CD34+ +Primary dendritic cells, + Eosinophils Monocytes + + OsteoblastsKeratinocyte + + Stromal endometrial cells Stromal cell TF274

[0734] As shown in the table, DR3 and two forms of TNF-gamma aredifferentially expressed in different tissues and cells. In thelibraries tested, DR3 was found to be expressed in most tissues, inactivated T-cells, monocytes, dendritic cells, TH2 cells, and severalother cell lines (such as U937, HeLa) and tumor tissues (such ashepatocellular tumor and Hodgkin's lymphoma). DR3 expression wasincreased in LNCAP prostate carcinoma cell line treated with 30 nM ofsynthetic androgen. TNF-gamma-alpha is only expressed in a few tissuesor cells such as fetal brain, fetal heart, adipose, kidney cortex,olfactory epithelium, pancreatic carcinoma and HUVEC. In contrast,TNF-gamma-beta has a much broader expression pattern. At the cellularlevel, only endothelial cell, activated T-cells, monocytes,keratinocytes, HeLa and Jurkat cells express TNF-gamma-beta. Only HUVEC,fetal brain, and fetal heart cDNA libraries express both forms ofTNF-gamma and DR3. TNF-gamma-alpha, TNF-gamma-beta, and DR3 are notexpressed in resting T-cells or early stage of activated T-cells (12hr). DR3 becomes detectable at 16 hr, and both DR3 and TNF-gamma-betabecome detectable in T-cells at 24 hr after PHA stimulation. Thetime-dependent induction of DR3 and then TNF-gamma-beta in activatedT-cells suggest that DR3 and TNF-gamma may play an import role inactivation induced apoptosis.

[0735] Northern blot and cDNA database analysis indicated that DR3expression is found predominantly in tissues with high content oflymphocytes, TNF-gamma is predominantly expressed in endothelial cells,monocytes and activated T-cells. Thus, DR3 and TNF-gamma may be involvedin the activation-induced apoptosis and the negative selection oflymphocytes. The expression pattern of DR3, TNF-gamma-alpha, andTNF-gamma-beta by different cells and tissues. Expression of differentsplicing variant forms of DR3 or TNF-gamma is likely to set the balancebetween susceptibility and protection from DR3-mediated apoptosis. It isclear that the pathway leading to apoptosis is highly regulated processand involving a series of proteins.

[0736] Another ligand for DR3, named as Apo3L has been describedrecently, which was also published as Tweak. Unlike TNF-gamma,Apo-3L/Tweak expressed in a wide variety of tissues. Theinterrelationship and functional importance between these two DR3ligands remain to be investigated.

Conclusion

[0737] One pair of novel receptor and ligand of TNF superfamily, DR3 andTNF-gamma, has been identified. Unlike other ligands of TNF family,TNF-gamma exists in two different forms and is differentially expressedin different cells and tissues. It has been suggested that one of themechanisms for regulating DR3 function is through alternative splicingof DR3. Alternative pre-mRNA splicing generates at least 11 isoforms ofDR3, providing a range of functional outcomes that may help shape theimmune response. Our data suggested that DR3 function can also beregulated through alternative splicing and differentially expression ofits ligand, TNF-gamma. These findings have great impact on how we viewthe regulation of apoptosis and TNF receptor superfamily function.Identification of two differentially expressed DR3 ligand variantsraised the possibility to selectively modulate apoptosis, immuneresponse and immune surveillance of tumor. Further characterization ofphysiological and pathological function of two differentially expressedTNF-gamma may provide new insights into the biological activities andphysiological function as well as therapeutic application of TNFreceptor and ligand superfamily. Understanding the role and mechanismsof action of these genes should allow us to develop ways to regulateapoptosis and cell proliferation in a variety of physiological andpathological conditions.

Materials and Methods Apoptosis Assay

[0738] Bovine pulmonary artery endothelia cells (BPAEC) were incubatedwith various concentrations of TNF-gamma for 48 hours. Apoptosis wasassessed morphologically and by nuclear staining with Hoechst 33342fluorescence dye (10 mg/ml) in triplicate. Live and apoptotic cells werescored in four random fields, about 1,000 cells were counted. The DNAfragmentation was analyzed as described previously.

BIAcore Receptor-ligand Binding Assay

[0739] Generation of recombinant receptor DR3-Fc fusion protein andrecombinant TNF-gamma were described in previous papers. PurifiedTNF-gamma or DR3-Fc was immobilized on BIAcore respectively. PurifiedDR3-Fc or TNF-gamma was analyzed on a BIAcore instrument flowcellderivatized with TNF-gamma or DR3-Fc. The net bound (off-rate) region ofthe plot after binding of TNF-gamma to immobilized DR3-Fc receptor, orbinding of DR3-Fc to immobilized TNF-gamma, was measured in relativemass units (RU) versus time. The binding conditions were performed athigh receptor chip densities under diffusion-limited conditions.

Co-Immunoprecipitation and Western Blot Analysis

[0740] Polyclonal antisera against TNF-gamma were prepared in rabbits asdescribed previously (Ni, J., et al., J. Biol. Chem. 272:10853-10858,(1997)). The Fc-extracellular domains of DR3 or Fc alone and thecorresponding ligands were prepared and binding assays were performed asdescribed elsewhere. The respective Fc-fusions were precipitated withprotein G-Sepharose and co-precipitated soluble ligands were detected byimmunoblotting with anti-TNF-gamma antibody. The samples were loadedinto a gel [NOVEX Pre-Cast Gels] (4˜20% Tris-Glycine Gel). Blotting anddetection was performed as described in BM Chemiluminescence WesternBlotting kit protocol.

FACS Analysis

[0741] Cells were collected by trypsinization or aspiration, andcentrifuged at 1500-2000 rpm for 5 min. The cell pellets wereresuspended and washed in 5 ml ice-cold PBS twice. The cells wereincubated for 30 min at 40° C. with antibody (10 mg/ml ) to TNF-gamma todetected expression of TNF-gamma on cell surface, with DR3-Fc or LTbR-Fc(10 mg/ml) for receptor and ligand binding in the binding buffer (HBSScontaining 10% BSA, 20 mM HEPES, pH 7.2, 0.02% NaN3). Purified human IgG(25 mg/ml) was used as a control. Cells were then washed and stainedwith phycoerythrin (PE) conjugated to goat anti-rabbit or anti-human IgGat 20 mg/ml. Fluorescence was analyzed by a FACscan flow cytometer(Becton Dickinson, Mountain View, Calif.).

NF-kappaB-SEAP (Secreted Alkaline Phosphatase) Reporter Assay

[0742] U937 cells were transfected using lipofectamine (followingmanufacturer's instructions) with 0.2 mg of reporter plasmid(NF-kappaB-SEAP). The transfected U937 cells were collected and added tothe 96-well plate (200 ml/well) with various concentration of activeTNF-gamma or inactivated (boiled) TNF-gamma or in combination withvarious concentration DR3-Fc receptor and 100 ng/ml of TNF-gamma. AfterIncubation at 37° C. for 72 hr, the NF-kappaB activity was measured withluminometer at absorbance of 450 nm.

Tissue and Cell Distribution Analysis Using PCR on a Large Collection ofcDNA Libraries and cDNA Database

[0743] To study the tissue distribution of DR3, TNF-gamma-alpha andTNF-gamma-beta, two gene specific primers were synthesized for eachgene. Over 100 cDNA libraries are tested and the libraries gave apositive predicted size signal are indicated as +.

In vivo Tumorigenicity Assay

[0744] The full length TNF-gamma and extracellular domain of DR3 wascloned into pcDNA3 expression vector (Invitrogen, Carlsbad, Calif.) andtransfected to MCA 38 cells, respectively. Subsequent to transfection,G418 selection, and cloning, three clones from each constructs werepicked for tumorigenicity study. The expression of TNF-gamma and DR3 inMCA 38 cells were confirmed by Northern analysis. MCA 38 cells (1×10⁶cells/mouse) expressing TNF-gamma or DR3 extracellular domain wereinjected into C57BL6/6 mice. Mice then were randomized and tumors weremeasured twice weekly. The tumor size was assessed by measuringperpendicular diameters with a caliper and calculated by multiplying themeasurements of diameters in two dimensions. Data are represented as themean+/−SD of 6 mice in each group.

Example 15 TNF-gamma-alpha, a Novel Member of TNF Cytokine Family,Causes Endothelial Cell Apoptosis Background

[0745] TNF-gamma-alpha is a novel protein with a molecular weight of 22kD that was recently identified by searching the Human Genome Sciences(HGS) cDNA database (Tan, K. B., et al, Gene 204:35-46 (1997)).TNF-gamma-alpha is a type II membrane protein and exhibits about 30%sequence homology to human tumor necrosis factor alpha (TNFalpha). Thisnewly identified member of the TNF family has been demonstrated to beabundantly expressed in endothelial cells as well as in kidney, lung andprostate. TNF-gamma-alpha expression in HL-60 and THP1 cells was inducedby PMA treatment. Radiation hybrid mapping localized TNF-gamma gene onchromosome 9q32, near CD30L. Because of its overexpression inendothelial cells, TNF-gamma-alpha has been suggested to possibly play arole in vascular functions (Tan, K. B., et al, Gene 204:35-46). Thepresent study was undertaken to explore whether TNF-gamma-alpha inducesendothelial cell apoptosis, a phenomenon suggested to be one cause ofendothelial cell damage contributing to various inflammatory disordersand cardiovascular dysfunction (Bryant, D., et al, Circulation97:1375-1381 (1998)). To examine this possibility, we used bovinepulmonary artery endothelial cells (BPAEC) to which TNFalpha-inducedapoptosis has been demonstrated (Polunovsky, V. A., et al., Exp. CellRes. 214:584-594 (1994)). Apoptosis was detected on the basis ofmorphological (including ultrastructural) and biochemicalcharacteristics (DNA fragmentation). In addition, we studied the effectsof TNF-gamma-alpha on the activity of stress kinases, stress-activatedprotein kinase (SAPK/JNK) and p38 mitogen-activated protein kinase (p38MAPK), and the caspases. Both signaling pathways are believed to beimplicated in programmed cell death (Xia, Z., et al., Science270:1326-1331 (1995)). The expression of Fas and Bcl-2 inTNF-gamma-alpha-stimulated BPAEC was also determined in view of thedeath-promoting effect of Fas and the anti-apoptotic effect of Bcl-2(Nagata, S. and Golstein, P. Science 267:1449-1456 (1995)).

Materials and Methods Materials

[0746] TNF-gamma-alpha protein (22 kD) was provided by HGS. Ac-YVAD-AMCand Ac-DEVD-AMC were purchased from American Peptide (Sunnyvale, Calif.,USA). ZVAD-fmk and Ac-YVAD-CHO were obtained from Enzyme Systems(Dublin, Calif., USA) and Peptides International (Louisville, Ky., USA),respectively. Ac-DQMD-AMC, Ac-LEED-AMC, Ac-VETD-AMC and anti-p38 MAPKmAb were provided by SmithKline Beecham (SB) Pharmaceuticals (King ofPrussia, Pa., USA). Ac-IETD-AMC and mouse-anti-human JNK mAb werepurchased from Biomol Research Laboratories (Plymouth Meeting, Pa., USA)and PharMingen (San Diego, Calif., USA), respectively. Mouse soluble TNFreceptor 1(sTNFR1) and TNF receptor 2 (sTNFR2) was obtained from R&DSystems (Minneapolis, Minn., USA).

Cell Cultures

[0747] BPAEC were obtained from the American Type Culture Collection(Rockville, Md., USA). The cells were grown in DMEM supplemented with10% heat-inactivated FCS in a humidified environment of 5% CO₂/85% airat 37° C. as previously described (Yue, T. L., et al., Mol. Pharmacol.51:951-962 (1997)). Cells at a subconfluent density were used. Beforeexperiments, the medium was changed to DMEM contained 2% FCS. BPAEC frompassages 17-20 were used in all studies.

Morphological Assessment and Quantification of Apoptosis

[0748] To quantify cells undergoing apoptosis, cell monolayers werefixed and stained with Hoechst 33324 (Molecular probe, Eugene, Oreg.,USA) as described previously (Yue, T. L., et al., Mol. Pharmacol.51:951-962 (1997)). The morphological features of apoptosis (cellshrinkage, chromatin condensation, blebbing, and fragmentation) weremonitored by fluorescence microscopy. Transmission electron microscopystudy was done as reported previously (Yue, T. L., et al., Mol.Pharmacol. 51:951-962 (1997)).

DNA Fragmentation Analysis

[0749] DNA ladder: Cells treated with vehicle or TNF-gamma-alpha werelysed in lysis buffer containing 100 mM NaCl, 10 mM Tris-HCl, pH 8.0,2.5 mM EEDTA, 0.5% SDS, and 100 micrograms/ml protein kinase K. Thelysates were incubated at 55° C. for 16 h. After incubation, the lysateswere gently extracted three times with pheno/chloroform/isoamyl alcohol,precipitated in ethanol, treated with DNAse-free RNAse, re-extracted,and precipitated again as described previously. DNA electrophoresis wascarried out in 1.8% agarose gels containing ethidium bromide, and DNAfragmentations were visualized under ultraviolet light.

[0750] In situ end-labeling (TUNEL): BPAEC were cultured in two-chamberslides (Nunc) and treated with TNF-gamma-alpha for 8 to 24 h. In situdetection of apoptotic cells was performed by using terminaldeoxyribonucleotide transferase-mediated dUTP nick end labeling with anApopTag in situ apoptosis detection kit (Oncor) following themanufacturer's recommendation.

Stress-activated Protein Kinase (SAJPK/JNK) Assay

[0751] SAPK activity was measured using GST-c-Jun(₁₋₈₁) as bound toglutathione-Sepharose 4B as described previously (Yuc, T. L., et al.,Mol. Pharmacol. 51:951-962 (1997)). Briefly, the cells were treated withvehicle or TNF-gamma-alpha, washed, and lysed in lysis buffer. Thenuclear-free supernatant was normalized for protein content andimmuno-precipitated with anti-SAPK antibody-conjugated Sepharose beads.The mixture was rotated 4° C. for 3 h. The phosphorylation buffercontaining GST-c- Jun(₁₋₈₁) 10 μC[g-³² P1-ATP, 125 μM ATP and 100 mMMgCl, was added to the SAPK-bound beads in assay buffer. The reactionwas terminated after 20 min at 30° C. by addition of protein loadingbuffer and heated at 90° C. for 3 min. Phosphorylated proteins wereresolved in 10% SDA-polyacrylamide gel electrophoresis followed byautoradiography. The intensity of the bands was quantified byPhosphorImager (Yuc, T. L., et al., J. Mol. Cell. Cardiol. 30:495-507(1998)). cl p38 MAPK Assay

[0752] The cell lysates prepared as above were immuno-precipitated witanti-p38 MAKP antibody bound to protein A agarose for 4 h at 4° C. Thebeads were washed with lysis buffer and then with kinase buffer asdescribed previously (Kumar, S. M., et al., J. Biol. Chem.271:30864-30869 (1996)). The immune-complex kinase assay was initiatedby the addition of 25 μl of kinase buffer containing 2 μg of GST-ATF2and 50 micromolar [gamma-³²P] ATP (20 Ci/mmol). The phosphorylatedproducts were resolved by SDA-PAGE and visualized by Phosphorimager.

In Vitro Transfection of Dominant-interfering Mutant of c-JUN in BPAEC

[0753] The cells were plated in two-chamber slides. The cells werecotransfected with 0.5 μg/ml of Pegfp-c-1 (Clontech; Li, Y. and Horwitz,M. S. Biotechnology 23:1026-1028) as a fluorescent marker of transfectedcells together with 1 μg/ml of either the empty cloning vector pCDNA1(control) or the dominant-interfering c-Jun mutant pcDNA1-FlagΔ169 (Xia,Z., et al., Science 270:1326-1331 (1995)) using Calphos MaximizerTransfection Kit (Clontech) according to the manufacturer'srecommendation. Following transfection, the cells were allowed torecover in complete medium for 24 h. The cells were treated withTNF-gamma-alpha and the number of apoptotic cells was assessed bynuclear staining after fixation as described in Methods.

Caspase Activity Assay

[0754] The cells were treated with vehicle of TNF-gamma-alpha. Caspaseactivity assays were performed as reported previously (Yuc, T. L., etal., supra). Briefly, cells were harvested and suspended in buffercontaining 25 mM HEPES, pH 7.5, 10% sucrose, 0.1% CHAPS, 2 mM DDT, 5 mMPMSF, and 1 μM pepstatin A. The suspension was forced through a 25 gaugeneedle 10 times to break cells. the homogenate was centrifuged at100,000×g for 1 h, and the cleared lysates were used for enzyme assays.Cell extracts (5-20 μg protein) were diluted into the assay buffer(Table 2) and preincubated for 10 min at 30 ° C. prior to the additionof the substrates. Levels of released 7-amino-4-methylcocmarin (AMC)were measured with a Cytofluor-4000 fluorescent plate reader (PerseptiveBiosystems) at an excitation and emission wavelengths of 360 nm and 460nm, respectively.

Immunohistochemical Analysis for Fas, Bcl-2 and Caspase-3 Expression

[0755] The cells were cultured in two-chamber slides. After treatmentwith vehicle or TNF-gamma-alpha, the cells were fixed with 4%paraformaldehyde for 30 min at 4° C. and then changed to cold PBS. Thecells were treated with 0.2% Triton X-100 for 40 min at 4° C., washedwith cold PBS and then non-specific immunoglobulin binding sites wereblocked with normal goat serum (Vector Laboratories) for 1 h at roomtemperature. The cell samples were incubated with the primary antibodymouse anti-human Fas (Upstate Biotechnology), mouse anti-human Bcl-2(DAKO) or rabbit anti-human CPP32 p17 peptide polyclonal antisera(SmithKline Beecham), for 1 h at room temperature. As a negativecontrol, the cell samples were incubated with nonimmune IgG (for Bcl-2and CPP32) or IgM (for Fas) instead of the primary antibody. Afterincubation with the primary antibody, cells were washed with PBS andthen incubated for 30 min with a secondary antibody conjugated tofluorescein isothiocyanate. Cells were washed, treated with Veetashieldmounting medium (Vector Laboratories) and viewed by fluorescencemicroscopy (Olympus IX70).

Statistical Analysis

[0756] All values are represented as mean±S.E.M. of n independentexperiments. Statistical evaluation was performed by using one-wayanalysis of variance. Differences with a value of p<0.05 were consideredsignificant.

Results TNF-gamma-alpha Induces Apoptosis in BPAEC

[0757] When BPAEC were exposed to TNF-gamma-alpha the cells shrunk andretracted from their neighboring cells, and the cytoplasma becamecondensed. Cells stained with Hoechst 33324 and assessed by fluorescencemicroscopy demonstrated condensed chromatin of fragmented nuclei andblebbing of the plasma membrane. The study with transmission electronmicroscopy showed that TNF-gamma-alpha-treated BPAEC contained manycells undergoing morphologic alterations characteristic of apoptosisincluding condensation of chromatin and appearance of apoptotic bodies.The characteristic degradation of DNA into oligonucleosomal-lengthfragmentation was observed when the cells were exposed toTNF-gamma-alpha (30-300 ng/ml) for 24 h. The DNA fragments in situ wasfurther visualized by using TUNEL method. A considerable fraction ofendothelial cells treated with TNF-gamma-alpha showed positive staining;no positively stained cells were found in the vehicle-treated cultures.

[0758] TNF-gamma-alpha-induced endothelial cell apoptosis was a time-and concentration-dependent process with an EC₃₀ value of 72 ng/ml. Asignificant increase in the number of cells with apoptotic morphologicalchanges was apparent 6-8 h after exposure of the cells toTNF-gamma-alpha. Under similar conditions, TNF-alpha at 10 ng/ml inducedapoptosis in PEAPC by 16.7±3.2% (n=4).

Effects of sTNFR1 and sTNFR2 on TNF-gamma-alpha-induced Apoptosis inBPAEC

[0759] Neither sTNFR1 nor sTNFR2 showed effect onTNF-gamma-alpha-induced apoptosis in BPAEC. Under the same conditionTNFa-induced apoptosis in BPAHC was reduced by sTNFR1 significantly.

[0760] Regulation of Fas and Bcl-2 Expression in Endothelial Cells byTNF-gamma-alpha. Immunocytochemical analysis of Fas and Bcl-2 proteinswas determined at 8 and 24 h after treatment with TNF-gamma-alpha. Thebasal level of Fas in BPAEC was undetectable. However, a significantnumber of cells expressing Fas receptor were detected at 8 and 24 hafter stimulation. When mouse IgM was substituted for the primaryantibody, positive Fas immunoreactivity was not detected. In contrast,Bcl-2 expression was not detected in neither unstimulated norTNF-gamma-alpha-treated BPAEC.

Activation of SAPK/JNK and p-38 MAPK

[0761] With regard to the effects of TNF-gamma-alpha on SAPK/JNKactivity in BPAEC, exposure of endothelial cells to TNF-gamma-alphainduced a rapid activation of SAPK/JNK. A significant increase inSAPK/JNK activity was detected 20 min after stimulation, peaked at 40min. and then returned to the basal levels after 60 min.TNF-gamma-alpha-induced activation of SAPK/JNK in endothelial cells is aconcentration-dependent process. Some basal activities of SAPK/JNKactivity was increased by 5.6±1.4 folds (p<0.05 n=4) and 9.1±1.8 folds(p<0.01 n=6) level in the presence of 50 and 300 ng/ml ofTNF-gamma-alpha, respectively. TNF-gamma-alpha activated p38 MAPK inBPAEC with a similar time course as SAPK/JNK but to a lesser extent. Thepeak of p38 MAKP activity was increased by 3.1+0.5 and 3.8±0.4 foldsover the basal level in the presence of 100 and 300 ng/ml ofTNF-gamma-alpha, respectively.

Effects on TNF-gamma-alpha-induced Apoptosis by Expression ofDominant-interfering mutant of c-JUN in BPAEC or by the p38 MAPKInhibitor, SB203580

[0762] To investigate the role of SAPK/JNK in TNF-gamma-alpha-inducedapoptosis in BPAEC, we transfected BPAEC with a dominant-interferingmutant of c-JUN, pCDNA1-FlagΔ169, in which a deletion in theNH2-terminal transactivation domain that includes the binding site forJNK (Xia, Z., et al., supra). Expression of dominant-interfering c-JUNconstruct in BPAEC reduced TNF-gamma-alpha-induced apoptosis by 62.8%(p<0.05). TNF-gamma-alpha-induced apoptosis in BPAEC was also attenuatedby a specific p38 MAPK inhibitor, SB203580, in a concentration dependentmanner. In the presence of 3 and 10 μM of SB203580,TNF-gamma-alpha-induced BPAEC apoptosis was reduced by 33% (p<0.05) and51% (p<0.01), respectively. No further inhibition was observed when theconcentration of SB203580 was increased.

Activation of Caspases in BPAEC by TNF-gamma-alpha

[0763] TNF-gamma-alpha-induced BPAEC apoptosis was attenuated byZVAD-fmk, an irreversible cell-permeable inhibitor of caspase (Jocobson,N. L., et al., Cell Biol. 133:1041-1051 (1996)), added to the culturemedium 1 h prior to TNF-gamma-alpha treatment. Under the sameconditions, the addition of Ac-YYAD-CHO, a relatively specific inhibitorof caspase-1 (Thorberry, N. A., et al., Nature (Lond) 356:768-774(1992)), up to 100 μM showed no effect in enhancing BPAEC rescue. Tofurther determine which of the caspase family members are activated inthe TNF-gamma-alpha-induced apoptotic process in the endothelial cells,we examined cell extracts for proteolytic activity. The relative ratesof AMC formation were measured with a series of defined peptide sequencevariants that are relatively specific for caspase 1, 3, 4, 7, or 8 underthe optimal conditions as described previously (Yuc, T. L., et al.,supra). Similar results were observed from three repeated experiments.Cell extracts from TNF-gamma-alpha-treated BPAEC were highly active onAc-DEVD-AMC and to a lesser extent on Ac-DQMD-AMC, but not active on theremaining three substrates which are more specific for caspase 1, 4, and8. The proteolytic activity appeared at 6 h after the cells were treatedwith TNF-gamma-alpha, peaked at 24 h, and gradually returned to basallevels within 48 h. The relative velocities of four substrate hydrolysisrates by the TNF-gamma-alpha-treated cell extracts and recombinantcaspase-3 were compared. The relative velocities of the two enzymesources of four substrates were very similar.

[0764] To further confirm that caspase-3 is activated by TNF-gamma-alphain BPAEC, immunocytochemical detection of its enzymatically active form,the 17-kD subunit, was performed. The antibody was raised against apeptide from the C-terminal portion of the p17 subunit. The neoepitopeantibody only binds caspase-3 if there has been specific cleavagebetween the “p-10” and “p-20” subunits. Using this neoepitope antibody,only processed caspase-3 is detected, but not the porenzyme (Yuc, T. L.,et al., supra). The 17 kD subunit of caspase-3 was detected inTNF-gamma-alpha-treated but not vehicle-treated BPAEC, and was localizedwith fragmented nuclei within the cells.

Discussion

[0765] The studies presented in this paper demonstrate thatTNF-gamma-alpha, a novel TNF-like cytokine and a type II transmembraneprotein, induces intensive apoptosis in cultured endothelial cells asreflected by morphological and biochemical criteria. Under ourexperimental conditions, spontaneous BPAEC death was approximately 2-4%which is in accord with a previous observation (Polunovsky, V. A., etal., supra). The effect of TNF-gamma-alpha was concentration-dependentwith an EC₈₀ value of 72 ng/ml (3.5 nM) and a significant number ofapoptotic cells was detected 6-8 h after treatment. Moreover, theexpression of pro-apoptotic gene, Fas, was demonstrated inTNF-gamma-alpha-treated BPAEC, which is consistent with that observed inapoptotic endothelial cells reported previously (Yuc, T. L., et al.,supra).

[0766] The receptor(s) mediating TNF-gamma-alpha activity has not beenidentified as yet. To examine whether TNF-gamma-alpha acts via distinctreceptor(s), we tested the effects of sTNFR1 and sTNFR2 onTNF-gamma-alpha-induced apoptosis in BPAEC. These two TNFRs have beenshown previously to block the cell surface TNFR1 and TNFR2 mediated TNFbioactivities on responsive cell lines (data from R&D Systems). NeithersTNFR1 nor sTNFR2 inhibited the effect of TNF-gamma-alpha on BPAEC. Incontrast, TNFa-induced apoptosis in BPAEC was significantly reduced bysTNF1. The results suggest clearly that TNF-gamma-alpha-induced celldeath is independent of sTNFR1 or TNFR2.

[0767] Recent research efforts on TNF family members have demonstratedthat TNFa and Fas activate stress protein kinases, SAPK/JNK and p38MAPK, in a variety of cell types (Sluss, H. K., et al., Cell Biol.14:8376-8384 (1994)), however, the effects of other members of thisfamily on SAPK and p38 MAPK are not well studies. Moreover,controversies regarding the role of SAPK/JNK and p38 MAPK in TNFa orFas-mediated cell death have been reported. For example, TNFa-inducedapoptosis is dependent on JNK activity in U937 cells (Verjeij, M., etal., Nature (Lond) 380:75-79 (1995); Zanke, B. W., et al., Curr. Biol.6:606-613 (1996)) but not in fibroblasts (Reinhard, C., et al., EMBO J.16:1080-1092 (1997)) indicating that the consequences of JNK activationvary considerably among cell types. Fas-mediated JNK activation occurswith a different kinetics from that of TNFa, suggesting that TNFa andFas most likely activate JNK through a different mechanism (Wilson, D.J., et al., Eur. J. Immunol. 26:989-994 (1996)). Moreover, Juo, et al.,reported recently that blockade of p38 MAPK by a specific p38 MAPKinhibitor did not affect Fas-mediated apoptosis in Jurkat cells (Juo,P., et al., Mol. Cell Biol. 17:24-35 (1997)). Therefore, we wereinterested in finding whether TNF-gamma-alpha activates JNK and p38MAPK, and what is the role of this activation inTNF-gamma-alpha-mediated apoptosis in BPAEC. The present investigationclearly demonstrates that both JNK and p38 MAPK were rapidly activatedby TNF-gamma-alpha in a similar fashion as observed in TNFa-activatedU937. Moreover, expression of dominant-interfering mutant of c-JUN inBPAEC reduced TNF-gamma-alpha-induced cell death indicating thatTNF-gamma-alpha-induced apoptosis in BPAEC was dependent on JNKactivity. To address the potential involvement of p38 MAPK inTNF-gamma-alpha-mediated apoptosis in BPAEC, a specific p38 MAPKinhibitor SB203580 was tested. This inhibitor has been shown tospecifically inhibit p38 MAPK activity in vitro with no effect on avariety of kinases tested, including JNK and ERK-1 (Cuenda, A., et al.,FEBS Lett. 364:229-233 (1995)). TNF-gamma-alpha-induced apoptosis inBPAEC was also reduced by SB203580 in a concentration-dependent manner,indicating that p38 MAPK signaling pathway is involved inTNF-gamma-alpha-mediated BPAEC apoptosis. This effect is different fromthat observed in Fas-mediated apoptosis in Jurkat cells in whichSB203580 had no protective effect (Juo, P., et al., supra). Moreover,TNF-gamma-alpha-induced p38 MAPK activation occurs with must fasterkinetics in BPAEC than that observed in Jurkat cells in which the peakof p38 MAPK activation was at 2-4 h after stimulation by Fas, indicatingTNF-gamma-alpha and Fas most likely activate p38 MAPK through adifferent mechanism with a different outcome. Our data further suggeststhat different members of TNF family may have different signalingpathways to mediate cell death or have different effects in differentcell types.

[0768] Recent work has supported a central role for the caspase familymembers, as effectors of apoptosis (Kumar, S. M., et al., supra).However, the role of caspases in endothelial cell apoptosis has not beensufficiently explored. Two characteristic features of the caspase familyhave been elucidated; they cleave their target proteins after specificaspartic acids, resulting in two subunits that together form the activesite of the enzyme (Nicholson, D. W., et al., Nature (Lond) 376:37-43(1995); Kumar, S. M., et al., supra). Among the caspase family,caspase-3 (CPP32) has been considered as a central component of theproteolytic cascade during apoptosis and plays a key role in this family(Wang, X., EMBO J. 15:1012-1020 (1996); Woo, M., et al., GeneDevelopment 12:806-819 (1998)). TNF-gamma-alpha-induced BPAEC apoptosiswas inhibited by ZVAD-fmk, indicating a potential role for the caspasefamily in this effector pathway for apoptosis. To determine which of thecaspase family members are involved, we examined the substratespecificity of proteolytic activity in the extracts fromTNF-gamma-alpha-activated BPAEC by measuring the relative rate of AMCformation from 6 different substrates which are relatively specific forcaspases 1, 3, 4, 7 and 8 (Talanian, R. V., et al., J. Biol. Chem.272:9677-9682 (1997)). Treatment of BPAEC with TNF-gamma-alpha resultedin a significant increase in proteolytic activity towards DEVD-AMCmainly and DQMD-AMC to some extent, both of which show the relativespecificity for caspase-3 (Kumar, S. M., et al., supra). There was noinduction in proteolytic activity in TNF-gamma-alpha-activated cellextracts when Ac-YVAD-AMC, LEED-AMC or VETD-AMC were used as thesubstrate, indicating that caspases 1, 4 and 8 might not be involved.Moreover, comparison of the substrate specificity of the extracts fromTNF-gamma-alpha-treated BPAEC with recombinant caspase-3 showed asimilar pattern, further suggesting that caspase-3 may be thepredominant member in the caspase family activated by TNF-gamma-alpha.Furthermore, immunocytochemical studies detected the active form ofcaspase-3 in TNF-gamma-alpha treated BPAEC. It was reported thatmultiple caspase homologues were found in both the cytoplasm and nucleusin etoposide-induced apoptosis in HL-60 cells (Martins, I. M., et al.,J. Biol. Chem. 272:7421-7430 (1997)). Interestingly, inTNF-gamma-alpha-induced apoptotic BPAEC the immunoreactive 17kD subunitof caspase-3 was only localized with fragmented nuclei, furtherindicating a role of caspase-3 in TNF-gamma-alpha-induced apoptosis.Whether this active caspase-3 was transported into the nucleus or theinactive caspase-3 is already in the nucleus awaiting activationpromoted by TNF-gamma-alpha requires further investigation. Takentogether, these results suggest that caspase-3 was activated byTNF-gamma-alpha-induced cell apoptosis. However, our results cannotexclude other members of this family, especially those closely relatedto caspase-3, such as caspase-7, in mediating TNF-gamma-alpha-inducedapoptosis. Moreover, ZVAD-fmk was less effective at the later time (30h) compared to the earlier time (14 h) for inhibitingTNF-gamma-alpha-included apoptosis in BPAEC, suggesting acaspase-independent of negative-feedback mechanism may exist at thelater phase of TNF-gamma-alpha-induced BPAEC apoptosis.

[0769] In summary, the present studies have demonstrated thatTNF-gamma-alpha, a novel member of TNF cytokine family, causesendothelial cell apoptosis. TNF-gamma-alpha appears to act through areceptor which is distinct from TNF receptors 1 or 2. The effect ofTNF-gamma-alpha is via activation of the stress protein kinases,SAPK/JNK and p38 MAPK., and the caspases, mainly caspase-3 likeprotease. Apoptotic programmed cell death has been suggested to be acause of endothelial cell damage contributing to various inflammatorydisorders and cardiovascular injury (Karsan, A. Trends Cardiovasc. Med.8:19-24 (1998)). Moreover, endothelial cell apoptosis may be animportant mechanism involved in a balance between antiangiogenic andproangiogenic processes, and loss of this balance will lead to a varietyof diseases such as solid tumor metastasis and retinopathy (Folkman, J.and Shing, J. J. Biol. Chem. 267:10931-10934 (1992); Brooks, P. C., etal., Cell 79:1157-1164 (1994)).

Example 16 Protein Fusions of TNF-gamma Alpha or TNF-gamma-beta

[0770] TNF-gamma alpha or TNF-gamma-beta polypeptides of the inventionare optionally fused to other proteins. These fusion proteins can beused for a variety of applications. For example, fusion of TNF-gammaalpha or TNF-gamma-beta polypeptides to His-tag, HA-tag, protein A, IgGdomains, FLAG, and maltose binding protein facilitates purification.(See EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).)Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflifetime in vivo. Nuclear localization signals fused to TNF-gamma alpha orTNF-gamma-beta polypeptides can target the protein to a specificsubcellular localization, while covalent heterodimer or homodimers canincrease or decrease the activity of a fusion protein. Fusion proteinscan also create chimeric molecules having more than one function.Finally, fusion proteins can increase solubility and/or stability of thefused protein compared to the non-fused protein.

[0771] In one embodiment, TNF-gamma-alpha or TNF-gamma-betapolynucleotides of the invention are fused to a polynucleotide encodinga “FLAG” polypeptide. Thus, a TNF-gamma-alpha-FLAG or aTNF-gamma-beta-FLAG fusion protein is encompassed by the presentinvention. The FLAG antigenic polypeptide may be fused to aTNF-gamma-alpha or TNF-gamma-beta polypeptide of the invention at eitheror both the amino or the carboxy terminus. In preferred embodiments, aTNF-gamma-alpha-FLAG or a TNF-gamma-beta-FLAG fusion protein isexpressed from a pFLAG-CMV-5a or a pFLAG-CMV-1 expression vector(available from Sigma, St. Louis, Mo., USA). See, Andersson, S., et al.,J. Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et al., Proc. Natl.Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature 308:241 (1984)(each of which is hereby incorporated by reference). In furtherpreferred embodiments, a TNF-gamma-alpha-FLAG or a TNF-gamma-beta-FLAGfusion protein is detectable by anti-FLAG monoclonal antibodies (alsoavailable from Sigma).

[0772] In a specific embodiment, a TNF-gamma-beta-FLAG fusion proteinexpression construct was generated using the pFLAG-CMV-1 vector toexpress amino acid residues L-72 through L-251 of SEQ ID NO:20 fused toFLAG at the amino terminus.

[0773] In another specific embodiment, a TNF-gamma-beta-lacZ-FLAG fusionprotein expression construct was generated using the pFLAG-CMV-1 vectorto express amino acid residues L-72 through L-251 of SEQ ID NO:20 fusedto FLAG and lacZ at the amino terminus.

[0774] All of the types of fusion proteins described above can be madeusing techniques known in the art or by using or routinely modifying thefollowing protocol, which outlines the fusion of a polypeptide to an IgGmolecule.

[0775] Briefly, the human Fc portion of the IgG molecule can be PCRamplified, using primers that span the 5′ and 3′ ends of the sequencedescribed below. These primers also preferably contain convenientrestriction enzyme sites that will facilitate cloning into an expressionvector, preferably a mammalian expression vector.

[0776] For example, if the pC4 (Accession No. 209646) expression vectoris used, the human Fc portion can be ligated into the BamHI cloningsite. Note that the 3′ BamHI site should be destroyed. Next, the vectorcontaining the human Fc portion is re-restricted with BamHI, linearizingthe vector, and TNF-gamma alpha or TNF-gamma-beta polynucleotide,isolated by the PCR protocol described in Example 1, is ligated intothis BamHI site. Note that the polynucleotide is cloned without a stopcodon, otherwise a fusion protein will not be produced.

[0777] If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc Region

[0778]             GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAGAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT   (SEQ ID NO: 18)

Example 17 Assays to Detect Stimulation or Inhibition of B CellProliferation and Differentiation

[0779] Generation of functional humoral immune responses requires bothsoluble and cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL5, IL6,IL-7, IL10, IL-13, IL14 and IL15. Interestingly, these signals are bythemselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD 154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays that allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

Experimental Procedure

[0780] In Vitro assay- Purified TNF-gamma alpha or TNF-gamma-betaprotein, or truncated forms thereof, is assessed for its ability toinduce activation, proliferation, differentiation or inhibition and/ordeath in B-cell populations and their precursors. The activity ofTNF-gamma alpha or TNF-gamma-beta protein on purified human tonsillar Bcells, measured qualitatively over the dose range from 0.1 to 10,000ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay inwhich purified tonsillar B cells are cultured in the presence of eitherformalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilizedanti-human IgM antibody as the priming agent. Second signals such asIL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cellproliferation as measured by tritiated-thymidine incorporation. Novelsynergizing agents can be readily identified using this assay. The assayinvolves isolating human tonsillar B cells by magnetic bead (MACS)depletion of CD3-positive cells. The resulting cell population isgreater than 95% B cells as assessed by expression of CD45R (B220).Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M_ME, 100 U/ml penicillin, 10 microg/micro 1 streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of150 micro 1. Proliferation or inhibition is quantitated by a 20 h pulse(1 uCi/well) with ³H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

[0781] In Vivo assay- BALBIc mice are injected (i.p.) twice per day withbuffer only, or with 2 mg/Kg of TNF-gamma alpha or TNF-gamma-betaprotein, or truncated forms thereof. Mice receive this treatment for 4consecutive days, at which time they are sacrificed and various tissuesand serum collected for analyses. Comparison of H&E sections from normaland TNF-gamma alpha or TNF-gamma-beta protein-treated spleens identifythe results of the activity of TNF-gamma alpha or TNF-gamma-beta proteinon spleen cells, such as the diffusion of peri-arterial lymphaticsheaths, and/or significant increases in the nucleated cellularity ofthe red pulp regions, which may indicate the activation of thedifferentiation and proliferation of B-cell populations.Immunohistochemical studies using a B cell marker, anti-CD45R (B220),are used to determine whether any physiological changes to spleniccells, such as splenic disorganization, are due to increased B-cellrepresentation within loosely defined B-cell zones that infiltrateestablished T-cell regions.

[0782] Flow cytometric analyses of the spleens from TNF-gamma alpha orTNF-gamma-beta protein-treated mice is used to indicate whetherTNF-gamma alpha or TNF-gamma-beta protein specifically increases theproportion of ThB+, CD45R (B220) dull B cells over that which isobserved in control mice.

[0783] Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andTNF-gamma alpha or TNF-gamma-beta protein-treated mice.

[0784] The studies described in this example tested activity inTNF-gamma alpha or TNF-gamma-beta protein. However, one skilled in theart could easily modify the exemplified studies to test the activity ofTNF-gamma alpha or TNF-gamma-beta polynucleotides (e.g., gene therapy),agonists, and/or antagonists of TNF-gamma alpha or TNF-gamma-beta.

Example 18 T Cell Proliferation Assay

[0785] A CD3-induced proliferation assay is performed on PBMCs and ismeasured by the uptake of ³H-thymidine. The assay is performed asfollows. Ninety-six well plates are coated with 100 microliters/well ofmAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1)overnight at 4° C. (1 micrograms/ml in 0.05M bicarbonate buffer, pH9.5), then washed three times with PBS. PBMC are isolated by F/Hgradient centrifugation from human peripheral blood and added toquadruplicate wells (5×10⁴/well) of mAb coated plates in RPMI containing10% FCS and P/S in the presence of varying concentrations of TNF-gammaalpha or TNF-gamma-beta protein (total volume 200 microliters). Relevantprotein buffer and medium alone are controls. After 48 hours at 37° C.,plates are spun for 2 min. at 1000 rpm and 100 microliters ofsupernatant is removed and stored at −20° C. for measurement of IL-2 (orother cytokines) if an effect on proliferation is observed. Wells aresupplemented with 100 microliters of medium containing 0.5 microcuriesof ³H-thymidine and cultured at 37° C. for 18-24 hr. Wells are harvestedand incorporation of ³H-thymidine used as a measure of proliferation.Anti-CD3 alone is the positive control for proliferation. IL-2 (100U/ml) is also used as a control which enhances proliferation. Controlantibody which does not induce proliferation of T cells is used as thenegative controls for the effects of TNF-gamma alpha or TNF-gamma-betaproteins.

[0786] The studies described in this example tested activity inTNF-gamma alpha or TNF-gamma-beta protein. However, one skilled in theart could easily modify the exemplified studies to test the activity ofTNF-gamma alpha or TNF-gamma-beta polynucleotides (e.g., gene therapy),agonists, and/or antagonists of TNF-gamma alpha or TNF-gamma-beta.

Example 19 Effect of TNF-gamma Alpha or TNF-gamma-beta on the Expressionof MHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

[0787] Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such asTNF-alpha, causes a rapid change in surface phenotype (increasedexpression of MHC class I and II, costimulatory and adhesion molecules,downregulation of FCgammaRII, upregulation of CD83). These changescorrelate with increased antigen-presenting capacity and with functionalmaturation of the dendritic cells.

[0788] FACS analysis of surface antigens is performed as follows. Cellsare treated 1-3 days with increasing concentrations of TNF-gamma alphaor TNF-gamma-beta or LPS (positive control), washed with PBS containing1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilutionof appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutesat 4° C. After an additional wash, the labeled cells are analyzed byflow cytometry on a FACScan (Becton Dickinson).

Effect on the Production of Cytokines

[0789] Cytokines generated by dendritic cells, in particular IL-12, areimportant in the initiation of T-cell dependent immune responses. IL-12strongly influences the development of Thl helper T-cell immuneresponse, and induces cytotoxic T and NK cell function. An ELISA is usedto measure the IL-12 release as follows. Dendritic cells (10⁶/ml) aretreated with increasing concentrations of TNF-gamma alpha orTNF-gamma-beta for 24 hours. LPS (100 ng/ml) is added to the cellculture as positive control. Supernatants from the cell cultures arethen collected and analyzed for IL-12 content using commercial ELISA kit(e.g., R & D Systems (Minneapolis, Minn.)). The standard protocolsprovided with the kits are used.

Effect on the Expression of MHC Class II, Costimulatory and AdhesionMolecules

[0790] Three major families of cell surface antigens can be identifiedon monocytes: adhesion molecules, molecules involved in antigenpresentation, and Fc receptor. Modulation of the expression of MHC classII antigens and other costimulatory molecules, such as B7 and ICAM-1,may result in changes in the antigen presenting capacity of monocytesand ability to induce T cell activation. Increase expression of Fcreceptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

[0791] FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofTNF-gamma alpha or TNF-gamma-beta or LPS (positive control), washed withPBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodiesfor 30 minutes at 4° C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte Activation and/or Increased Survival

[0792] Assays for molecules that activate (or alternatively, inactivate)monocytes and/or increase monocyte survival (or alternatively, decreasemonocyte survival) are known in the art and may routinely be applied todetermine whether a molecule of the invention functions as an inhibitoror activator of monocytes. TNF-gamma alpha or TNF-gamma-beta, agonists,or antagonists of TNF-gamma alpha or TNF-gamma-beta can be screenedusing the three assays described below. For each of these assays,Peripheral blood mononuclear cells (PBMC) are purified from single donorleukopacks (American Red Cross, Baltimore, Md.) by centrifugationthrough a Histopaque gradient (Sigma). Monocytes are isolated from PBMCby counterflow centrifugal elutriation.

[0793] 1. Monocyte Survival Assay. Human peripheral blood monocytesprogressively lose viability when cultured in absence of serum or otherstimuli. Their death results from internally regulated process(apoptosis). Addition to the culture of activating factors, such asTNF-alpha dramatically improves cell survival and prevents DNAfragmentation. Propidium iodide (PI) staining is used to measureapoptosis as follows. Monocytes are cultured for 48 hours inpolypropylene tubes in serum-free medium (positive control), in thepresence of 100 ng/ml TNF-alpha (negative control), and in the presenceof varying concentrations of the compound to be tested. Cells aresuspended at a concentration of 2×10⁶/ml in PBS containing PI at a finalconcentration of 5 micrograms/ml, and then incubated at room temperaturefor 5 minutes before FACScan analysis. PI uptake has been demonstratedto correlate with DNA fragmentation in this experimental paradigm.

[0794] 2. Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of TNF-gamma alpha or TNF-gamma-beta andunder the same conditions, but in the absence of TNF-gamma alpha orTNF-gamma-beta. For IL-12 production, the cells are primed overnightwith IFN-gamma (100 U/ml) in presence of TNF-gamma alpha orTNF-gamma-beta. LPS (10 ng/ml) is then added. Conditioned media arecollected after 24 h and kept frozen until use. Measurement ofTNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commerciallyavailable ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) andapplying the standard protocols provided with the kit.

[0795] 3. Oxidative burst. Purified monocytes are plated in 96-wellplates at 2-1×10⁵ cell/well. Increasing concentrations of TNF-gammaalpha or TNF-gamma-beta are added to the wells in a total volume of 0.2ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After3 days incubation, the plates are centrifuged and the medium is removedfrom the wells. To the macrophage monolayers, 0.2 ml per well of phenolred solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, togetherwith the stimulant (200 nM PMA). The plates are incubated at 37° C. for2 hours and the reaction is stopped by adding 20 μl IN NaOH per well.The absorbance is read at 610 nm. To calculate the amount of H₂O₂produced by the macrophages, a standard curve of a H₂O₂ solution ofknown molarity is performed for each experiment.

[0796] The studies described in this example tested activity inTNF-gamma alpha or TNF-gamma-beta protein. However, one skilled in theart could easily modify the exemplified studies to test the activity ofTNF-gamma alpha or TNF-gamma-beta polynucleotides (e.g., gene therapy),agonists, and/or antagonists of TNF-gamma alpha or TNF-gamma-beta.

Example 20 Production of an Antibody

[0797] a) Hybridoma Technology

[0798] The antibodies of the present invention can be prepared by avariety of methods. (See, Current Protocols, Chapter 2.) As one exampleof such methods, cells expressing polypeptide(s) of the invention areadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation ofpolypeptide(s) of the invention is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity.

[0799] Monoclonal antibodies specific for polypeptide(s) of theinvention are prepared using hybridoma technology. (Kohler et al.,Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976);Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981)). In general, an animal (preferably a mouse) is immunized withpolypeptide(s) of the invention or, more preferably, with a secretedpolypeptide-expressing cell. Such polypeptide-expressing cells arecultured in any suitable tissue culture medium, preferably in Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

[0800] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981)). Thehybridoma cells obtained through such a selection are then assayed toidentify clones which secrete antibodies capable of binding thepolypeptide(s) of the invention.

[0801] Alternatively, additional antibodies capable of binding topolypeptide(s) of the invention can be produced in a two-step procedureusing anti-idiotypic antibodies. Such a method makes use of the factthat antibodies are themselves antigens, and therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, protein specific antibodies are used to immunize ananimal, preferably a mouse. The splenocytes of such an animal are thenused to produce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theprotein-specific antibody can be blocked by polypeptide(s) of theinvention. Such antibodies comprise anti-idiotypic antibodies to theprotein-specific antibody and are used to immunize an animal to induceformation of further protein-specific antibodies.

[0802] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed herein. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985).)

Isolation of Antibody Fragments Directed Against Polypeptide(s) from aLibrary of scFvs

[0803] Naturally occurring V-genes isolated from human PBLs areconstructed into a library of antibody fragments which containreactivities against polypeptide(s) of the invention to which the donormay or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793incorporated herein by reference in its entirety).

Rescue of the Library

[0804] A library of scFvs is constructed from the RNA of human PBLs asdescribed in PCT publication WO 92/01047. To rescue phage displayingantibody fragments, approximately 109 E. coli harboring the phagemid areused to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml ofampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Fiveml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×108 TUof delta gene 3 helper (M13 delta gene III, see PCT publication WO92/01047) are added and the culture incubated at 37° C. for 45 minuteswithout shaking and then at 37° C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 min. and the pelletresuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50ug/ml kanamycin and grown overnight. Phage are prepared as described inPCT publication WO 92/01047.

[0805] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)displaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990),resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

Panning of the Library

[0806] Immunotubes (Nunc) are coated overnight in PBS with 4 ml ofeither 100 μg/ml or 10 μg/ml of a polypeptide of the present invention.Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and thenwashed 3 times in PBS. Approximately 1013 TU of phage is applied to thetube and incubated for 30 minutes at room temperature tumbling on anover and under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under and over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage arethen used to infect 10 ml of mid-log E. coli TG1 by incubating elutedphage with bacteria for 30 minutes at 37° C. The E. coli are then platedon TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube-washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders

[0807] Eluted phage from the 3rd and 4th rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks, et al.,1991) from single colonies for assay. ELISAs are performed withmicrotitre plates coated with either 10 pg/ml of the polypeptide of thepresent invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISAare further characterized by PCR fingerprinting (see, e.g., PCTpublication WO 92/01047) and then by sequencing. These ELISA positiveclones may also be further characterized by techniques known in the art,such as, for example, epitope mapping, binding affinity, receptor signaltransduction, ability to block or competitively inhibit antibody/antigenbinding, and competitive agonistic or antagonistic activity.

Example 21 Method of Determining Alterations in the TNF-gamma Alpha orTNF-gamma-beta Gene

[0808] RNA is isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease). cDNA isthen generated from these RNA samples using protocols known in the art.(See, Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 secondsat 52-58° C.; and 60-120 seconds at 70° C., using buffer solutionsdescribed in Sidransky, D., et al., Science 252:706 (1991).

[0809] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofTNF-gamma alpha or TNF-gamma-beta are also determined and genomic PCRproducts analyzed to confirm the results. PCR products harboringsuspected mutations in TNF-gamma alpha or TNF-gamma-beta are then clonedand sequenced to validate the results of the direct sequencing.

[0810] PCR products of TNF-gamma alpha or TNF-gamma-beta are cloned intoT-tailed vectors as described in Holton, T. A. and Graham, M. W.,Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase(United States Biochemical). Affected individuals are identified bymutations in TNF-gamma alpha or TNF-gamma-beta not present in unaffectedindividuals.

[0811] Genomic rearrangements are also observed as a method ofdetermining alterations in the TNF-gamma alpha or TNF-gamma-beta gene.Genomic clones isolated using techniques known in the art arenick-translated with digoxigenindeoxy-uridine 5′-triphosphate(Boehringer Manheim), and FISH performed as described in Johnson, Cg. etal., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeledprobe is carried out using a vast excess of human cot-1 DNA for specifichybridization to the TNF-gamma alpha or TNF-gamma-beta genomic locus.

[0812] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of TNF-gamma alpha or TNF-gamma-beta(hybridized by the probe) are identified as insertions, deletions, andtranslocations. These TNF-gamma alpha or TNF-gamma-beta alterations areused as a diagnostic marker for an associated disease.

Example 22 Method of Detecting Abnormal Levels of TNF-gamma Alpha orTNF-gamma-beta in a Biological Sample

[0813] TNF-gamma alpha or TNF-gamma-beta polypeptides can be detected ina biological sample, and if an increased or decreased level of TNF-gammaalpha or TNF-gamma-beta is detected, this polypeptide is a marker for aparticular phenotype. Methods of detection are numerous, and thus, it isunderstood that one skilled in the art can modify the following assay tofit their particular needs.

[0814] For example, antibody-sandwich ELISAs are used to detectTNF-gamma alpha or TNF-gamma-beta in a sample, preferably a biologicalsample. Wells of a microtiter plate are coated with specific antibodiesto TNF-gamma alpha or TNF-gamma-beta, at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced using technique known in the art. The wells are blocked so thatnon-specific binding of TNF-gamma alpha or TNF-gamma-beta to the well isreduced.

[0815] The coated wells are then incubated for >2 hours at RT with asample containing TNF-gamma alpha or TNF-gamma-beta. Preferably, serialdilutions of the sample should be used to validate results. The platesare then washed three times with deionized or distilled water to removeunbounded TNF-gamma alpha or TNF-gamma-beta.

[0816] Next, 50 microliters of specific antibody-alkaline phosphataseconjugate, at a concentration of 25-400 ng, is added and incubated for 2hours at room temperature. The plates are again washed three times withdeionized or distilled water to remove unbounded conjugate.

[0817] 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution is then added to each well andincubated 1 hour at room temperature to allow cleavage of the substrateand flourescence. The flourescence is measured by a microtiter platereader. A standard curve is prepared using the experimental results fromserial dilutions of a control sample with the sample concentrationplotted on the X-axis (log scale) and fluorescence or absorbance on theY-axis (linear scale). The TNF-gamma alpha or TNF-gamma-beta polypeptideconcentration in a sample is then interpolated using the standard curvebased on the measured flourescence of that sample.

Example 23 Method of Treating Decreased Levels of TNF-gamma Alpha orTNF-gamma-beta

[0818] The present invention relates to a method for treating anindividual in need of a decreased level of TNF-gamma alpha orTNF-gamma-beta biological activity in the body comprising, administeringto such an individual a composition comprising a therapeuticallyeffective amount of TNF-gamma alpha or TNF-gamma-beta antagonist.Preferred antagonists for use in the present invention are TNF-gammaalpha or TNF-gamma-beta-specific antibodies.

[0819] Moreover, it will be appreciated that conditions caused by adecrease in the standard or normal expression level of TNF-gamma alphaor TNF-gamma-beta in an individual can be treated by administeringTNF-gamma alpha or TNF-gamma-beta, preferably in a soluble and/orsecreted form. Thus, the invention also provides a method of treatmentof an individual in need of an increased level of TNF-gamma alpha orTNF-gamma-beta polypeptide comprising administering to such anindividual a pharmaceutical composition comprising an amount ofTNF-gamma alpha or TNF-gamma-beta to increase the biological activitylevel of TNF-gamma alpha or TNF-gamma-beta in such an individual.

[0820] For example, a patient with decreased levels of TNF-gamma alphaor TNF-gamma-beta polypeptide receives a daily dose 0.1-100 mg/kg of thepolypeptide for six consecutive days. Preferably, the polypeptide is ina soluble and/or secreted form.

Example 24 Method of Treating Increased Levels of TNF-gamma Alpha orTNF-gamma-beta

[0821] The present invention also relates to a method for treating anindividual in need of an increased level of TNF-gamma alpha orTNF-gamma-beta biological activity in the body comprising administeringto such an individual a composition comprising a therapeuticallyeffective amount of TNF-gamma alpha or TNF-gamma-beta or an agonistthereof.

[0822] Antisense technology is used to inhibit production of TNF-gammaalpha or TNF-gamma-beta. This technology is one example of a method ofdecreasing levels of TNF-gamma alpha or TNF-gamma-beta polypeptide,preferably a soluble and/or secreted form, due to a variety ofetiologies, such as cancer.

[0823] For example, a patient diagnosed with abnormally increased levelsof TNF-gamma alpha or TNF-gamma-beta is administered intravenouslyantisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21days. This treatment is repeated after a 7-day rest period if the isdetermined to be well tolerated.

Example 25 Method of Treatment Using Gene Therapy—Ex Vivo

[0824] One method of gene therapy transplants fibroblasts, which arecapable of expressing soluble and/or mature TNF-gamma alpha orTNF-gamma-beta polypeptides, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37° C. for approximately one week.

[0825] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[0826] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0827] The cDNA encoding TNF-gamma alpha or TNF-gamma-beta can beamplified using PCR primers which correspond to the 5′ and 3′ endencoding sequences respectively. Preferably, the 5′ primer contains anEcoRI site and the 3′ primer includes a HindIII site. Equal quantitiesof the Moloney murine sarcoma virus linear backbone and the amplifiedEcoRI and HindIII fragment are added together, in the presence of T4 DNAligase. The resulting mixture is maintained under conditions appropriatefor ligation of the two fragments. The ligation mixture is then used totransform E. coli HB 101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted TNF-gamma alpha or TNF-gamma-beta.

[0828] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the TNF-gamma alpha or TNF-gamma-beta gene is thenadded to the media and the packaging cells transduced with the vector.The packaging cells now produce infectious viral particles containingthe TNF-gamma alpha or TNF-gamma-beta gene (the packaging cells are nowreferred to as producer cells).

[0829] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether TNF-gamma alpha or TNF-gamma-beta protein isproduced.

[0830] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

Example 26 Method of Treatment Using Gene Therapy—In Vivo

[0831] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) TNF-gamma alpha or TNF-gamma-betasequences into an animal to increase or decrease the expression of theTNF-gamma alpha or TNF-gamma-beta polypeptide. The TNF-gamma alpha orTNF-gamma-beta polynucleotide may be operatively linked to a promoter orany other genetic elements necessary for the expression of the TNF-gammaalpha or TNF-gamma-beta polypeptide by the target tissue. Such genetherapy and delivery techniques and methods are known in the art, see,for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622,5,705,151, 5,580,859; Tabata H. et al., Cardiovasc. Res. 35:470-479(1997); Chao J. et al., Pharmacol. Res. 35:517-522 (1997); Wolff J. A.Neuromuscul. Disord. 7:314-318 (1997); Schwartz B. et al,. Gene Ther.3:405-411 (1996); Tsurumi Y. et al., Circulation 94:3281-3290 (1996)(incorporated herein by reference).

[0832] The TNF-gamma alpha or TNF-gamma-beta polynucleotide constructsmay be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, intestine and the like). TheTNF-gamma alpha or TNF-gamma-beta polynucleotide constructs can bedelivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0833] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the TNF-gamma alpha or TNF-gamma-beta polynucleotidesmay also be delivered in liposome formulations (such as those taught inFelgner P. L. et al. Ann. NY Acad. Sci. 772:126-139 (1995), and AbdallahB. et al. Biol. Cell 85:1-7 (1995)) which can be prepared by methodswell known to those skilled in the art.

[0834] The TNF-gamma alpha or TNF-gamma-beta polynucleotide vectorconstructs used in the gene therapy method are preferably constructsthat will not integrate into the host genome nor will they containsequences that allow for replication. Any strong promoter known to thoseskilled in the art can be used for driving the expression of DNA. Unlikeother gene therapy techniques, one major advantage of introducing nakednucleic acid sequences into target cells is the transitory nature of thepolynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

[0835] The TNF-gamma alpha or TNF-gamma-beta polynucleotide constructcan be delivered to the interstitial space of tissues within an animal,including of muscle, skin, brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, eye, gland, and connective tissue. Interstitial space of thetissues comprises the intercellular fluid, mucopolysaccharide matrixamong the reticular fibers of organ tissues, elastic fibers in the wallsof vessels or chambers, collagen fibers of fibrous tissues, or that samematrix within connective tissue ensheathing muscle cells or in thelacunae of bone. It is similarly the space occupied by the plasma of thecirculation and the lymph fluid of the lymphatic channels. Delivery tothe interstitial space of muscle tissue is preferred for the reasonsdiscussed below. They may be conveniently delivered by injection intothe tissues comprising these cells. They are preferably delivered to andexpressed in persistent, non-dividing cells which are differentiated,although delivery and expression may be achieved in non-differentiatedor less completely differentiated cells, such as, for example, stemcells of blood or skin fibroblasts. In vivo muscle cells areparticularly competent in their ability to take up and expresspolynucleotides.

[0836] For the naked TNF-gamma alpha or TNF-gamma-beta polynucleotideinjection, an effective dosage amount of DNA or RNA will be in the rangeof from about 0.05 g/kg body weight to about 50 mg/kg body weight.Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kgand more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course,as the artisan of ordinary skill will appreciate, this dosage will varyaccording to the tissue site of injection. The appropriate and effectivedosage of nucleic acid sequence can readily be determined by those ofordinary skill in the art and may depend on the condition being treatedand the route of administration. The preferred route of administrationis by the parenteral route of injection into the interstitial space oftissues. However, other parenteral routes may also be used, such as,inhalation of an aerosol formulation particularly for delivery to lungsor bronchial tissues, throat or mucous membranes of the nose. Inaddition, naked TNF-gamma alpha or TNF-gamma-beta polynucleotideconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

[0837] The dose response effects of injected TNF-gamma alpha orTNF-gamma-beta polynucleotide in muscle in vivo are determined asfollows. Suitable TNF-gamma alpha or TNF-gamma-beta template DNA forproduction of mRNA coding for TNF-gamma alpha or TNF-gamma-betapolypeptide is prepared in accordance with a standard recombinant DNAmethodology. The template DNA, which may be either circular or linear,is either used as naked DNA or complexed with liposomes. The quadricepsmuscles of mice are then injected with various amounts of the templateDNA.

[0838] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The TNF-gamma alpha or TNF-gamma-beta template DNAis injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gaugeneedle over one minute, approximately 0.5 cm from the distal insertionsite of the muscle into the knee and about 0.2 cm deep. A suture isplaced over the injection site for future localization, and the skin isclosed with stainless steel clips.

[0839] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15micrometer cross-section of the individual quadriceps muscles ishistochemically stained for TNF-gamma alpha or TNF-gamma-beta proteinexpression. A time course for TNF-gamma alpha or TNF-gamma-beta proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence ofTNF-gamma alpha or TNF-gamma-beta DNA in muscle following injection maybe determined by Southern blot analysis after preparing total cellularDNA and HIRT supernatants from injected and control mice. The results ofthe above experimentation in mice can be use to extrapolate properdosages and other treatment parameters in humans and other animals usingTNF-gamma alpha or TNF-gamma-beta naked DNA.

Example 27 Gene Therapy Using Endogenous TNF-gamma Gene

[0840] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous TNF-gamma sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNumber WO 96/29411, published Sep. 26, 1996; International PublicationNumber WO 94112650, published Aug. 4, 1994; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989). This method involves the activation of a gene whichis present in the target cells, but which is not expressed in the cells,or is expressed at a lower level than desired. Polynucleotide constructsare made which contain a promoter and targeting sequences, which arehomologous to the 5′ non-coding sequence of endogenous TNF-gamma,flanking the promoter. The targeting sequence will be sufficiently nearthe 5′ end of TNF-gamma so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

[0841] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[0842] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[0843] Once the cells are transfected, homologous recombination willtake place which results in the promoter being operably linked to theendogenous TNF-gamma sequence. This results in the expression ofTNF-gamma in the cell. Expression may be detected by immunologicalstaining, or any other method known in the art.

[0844] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

[0845] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the TNF-gamma locus,plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII.The CMV promoter is amplified by PCR with an Xbal site on the 5′ end anda BamHI site on the 3′ end. Two TNF-gamma non-coding sequences areamplified via PCR: one TNF-gamma non-coding sequence (TNF-gammafragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′ end; the other TNF-gamma non-coding sequence (TNF-gammafragment 2) is amplified with a BamHI site at the 5′ end and a HindIIIsite at the 3′ end. The CMV promoter and TNF-gamma fragments aredigested with the appropriate enzymes (CMV promoter—Xbal and BamHI;TNF-gamma fragment 1—XbaI; TNF-gamma fragment 2—BamHI) and ligatedtogether. The resulting ligation product is digested with HindIII, andligated with the HindIII-digested pUC18 plasmid.

[0846] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×106cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0847] Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

[0848] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

Example 28 Analysis of Endothelial Cell Apoptosis by TNF-gamma-beta

[0849] Although this example is directed primarily towardsTNF-gamma-beta, one of ordinary skill in the art would immediatelyrecognize that the example may be performed essentially as described forany TNF-gamma protein of the invention (i.e., TNF-gamma-alpha and/orTNF-gamma-beta).

[0850] A. Caspase Assay.

[0851] An analysis of caspase activity was performed to gain insightinto possible mechanisms by which the anti-angiogenic activity ofTNF-gamma proteins suppress endothelial cell growth. Caspases are afamily of proteolytic enzymes that are activated under many apoptoticconditions. Caspases are a family of cysteine proteases that areactivated during the process of programmed cell death. In the responseto pro-apoptotic stimuli procaspases are proteolytically converted toactive enzymes. The amount of caspase enzymatic activity after treatmentwith TNF-gamma-beta was measured by determining the amount of cleavageof a chromophore- (pNA) linked caspase specific peptide substrate.

[0852] Using substrates specific for different caspases, it wasdetermined that TNF-gamma-beta activates Caspase 3. The substrate DEVD,which is activated by Caspase 3 and somewhat by other caspases, gave thegreatest fold induction by TNF-gamma-beta and was selected for activityassays. BAEC cells were treated with 0.0, 2.5, 5.0, 7.5, or 10.0micrograms/ml TNF-gamma-beta for 20 hours. Cell extracts were isolatedand samples of equal protein concentration were incubated with DEVD=pNApeptide substrate for 2 hours. Cleavage of the caspase substrate wasdetected by spectrophotometric analysis of duplicate cultures. Caspase 3activity was induced in a concentration dependent manner after treatmentof BAEC with TNF-gamma-beta.

[0853] An annexin/caspase activation analysis was performed to determinethe effect of two batches of TNF-gamma-beta proteins (designated “E1”and “E3”) on endothelial cell apoptosis. The experiments were performedessentially as follows. Briefly, bovine aortic endothelial cells(“BAEC”) were cultured in Clonetics growth medium (catalog numberCC-3121) EBM supplemented with catalog number CC-4143 EGM-MV(supplement). During periods of growth arrest, the cells were culturedin Growth Arrest Media (Human Endothelial SFM Basal Growth Medium, Gibcocatalog number 1111 1-044).

[0854] On day one, cells were plated in T75 culture flasks in culturemedium and incubated at 37° C. On day two, the culture medium wasaspirated and replaced with 10 ml of growth arrest medium (“GA medium”).The cultures were then incubated at 37° C. overnight. On day three, thefirst activity was to prepare treatments using two flasks of cells pertreatment. Aliquots of TNF-gamma-beta and control protein (TNF-alpha)were diluted in GA medium. Prior to adding proteins, 5 ml of media wereremoved so that the final volume in the flask is 5 ml. Cells were eitherleft untreated or treated with TNF-alpha (100 ng/ml, positive control),TNF-gamma-beta batch E1 (5 micrograms/ml) or TNF-gamma-beta batch E3 (5micrograms/ml). The cultures were then incubated in GA mediumsupplemented with either TNF-alpha or TNF-gamma-beta proteins for 20hrs.

[0855] On day four, cells were isolated for Annexin and cell extractswere analyzed for caspase essentially according to the followingprocedure. First, medium was transferred to labeled 15 ml conical vials.Flasks were washed two at a time with 3 ml/flask of PBS. All media andwashes were then pooled. 2 ml trypsin/EDTA was then added per flask andthe mixture was incubated for 1-2 minutes at 37° C. When all cellsbecame detached, the contents of each flask was transferred to itsappropriate tube. Tubes were then centrifuged in the Sorvall 6000Bcentrifuge for 7-10 minutes at setting #4.2. The supernatants were thencarefully aspirated and the cell pellets were resuspended in 1 ml pertreatment of 1×PBS with 0.01% BSA and the cells were counted.Approximately 500,000 cells were held in reserve for Annexin V analysis(see below).

[0856] For the caspase assay, the ApoTarget Caspase ColorimetricProtease Assay Sampler Kit (available from BioSource International,Inc., Catalog #KHZ1001) was used essentially according to themanufacturer's instructions. Briefly, the remaining cells were spundown, the supernatant was removed, and the cell pellet was resuspendedin 50 ml per 3×10⁶ cells of chilled lysis buffer. The cells were thenincubated on ice for 10 minutes then vortexed for 5 seconds. The tubeswere then centrifuged for 1 minute in a microcentrifuge (10,000×g). Thesupernatant (i.e., the cytosol extract) was then transferred to a freshtube and kept on ice. The protein concentrations were then determined bythe BCA method (Pierce).

[0857] The assay was set up in a 96 well microtitre U-bottom plate using50 micrograms of protein per sample in 50 microliters of lysis buffer.DTT was added to the 2×Reaction Buffer immediately before use (10 mMfinal concentration) and 50 microliters of 2×reaction buffer with DTTwas added to each reaction. Next, 5 ml of the 4 mM caspase substrates(200 mM final concentration) were added to each reaction and the tubeswere incubated at 37° C. for 1-2 hours, taking care to keep the samplesin the dark during incubation. The samples were then read at 405 nm in amicroplate reader. The no substrate values were subtracted and theresults plotted. The assay allows fold-increase in the activities ofcaspases 2, 3, 6, 8, or 9 by direct comparison to the level of theuninduced control.

[0858] This assay used two methods to examine the apoptotic actions ofTNF-gamma-beta E1 and E3 proteins. Annexin analysis showed an increasein a dead/dying population of cells locating at the midpoint of UL andLL in the TNF-alpha (positive control) and TNF-gamma-beta E1 samples.This population was not seen in untreated or TNF-gamma-beta E3 treatedsamples. Caspase analysis showed that TNF-alpha and TNF-gamma-beta E1(but not TNF-gamma-beta E3) induced a >10 fold increase in caspase 2 and3 activity, and a >5 fold increase in caspase 6 activity. There was asmaller increase in caspase 8 activity. Thus, both assays showedTNF-gamma-beta E1 but not TNF-gamma-beta E3 induced apoptosis in BAECs.

[0859] Further experiments have shown that TNF-gamma-beta E1 stimulatedcaspase 2 activity in a dose-dependent manner in BAECs. Also,TNF-gamma-beta E1 protein induced caspase 3 in BAEC, but caspase 3activity was not appreciably affected by TNF-gamma-beta E1 in eitherhuman microvascular endothelial cells (hMVEC) or in normal human dermalfibroblasts (NHDF). In addition, TNF-gamma-beta E3 protein stimulatedcaspase 3 activity in human aortic endothelial cells (hAEC).

[0860] A detailed caspase activation assay protocol is as follows.

Cell Culture

[0861] BAEC Culture Media: EGM-MV Bullet Kit (Clonetics Cat#CC-3125):(10% fetal bovine serum, 1 microgram/ml hydrocortisone, 10 ng/ml hEGF, 3ng/ml bFGF, and 10 micrograms/ml heparin, 12 ug/ml bovine brain extract,2×amphotericin B).

[0862] BAEC GA Media: Human Endothelial Serum Free Media, Gibco (Cat#11111-044), plus 5 ml of 100×penicillin/streptomycin, +0.1% FBS

[0863] Day One: Plate BAEC in 100 mm tissue culture dishes in culturemedia. Incubate ON at 37° C.

[0864] Day Two: Aspirate culture media and replace with 10 ml growtharrest (GA) media. Incubate at 37° C. overnight.

[0865] Day Three: Prepare treatments. Dilute proteins in GA media. Priorto adding proteins, remove appropriate volume of media so that the finalvolume per 100 mm dish is 2 ml.

Isolation of Cell Extracts

[0866] When incubation time is complete:

[0867] Transfer media to labeled 15 ml conical vials. Wash flasks with1.5 ml/dish of PBS, pooling all media/wash/trypsinized cells accordingto treatment. Add 2 ml per dish Trypsin/EDTA. Incubate 2-5 minutes at37° C. Check the cells under the microscope for progress in detaching.Rap the cells off the plate and, if necessary, scrape cells off theplate/flask with a cell lifter (Costar #3008). Transfer contents totheir appropriate tubes and centrifuge in the Sorvall 6000B for 7-10minutes at 2000 RPM.

[0868] Carefully aspirate the supernatants. Resuspend the pellets in 1ml PBS with 0.01% BSA. Transfer 10 microliters to a snap-cap Eppendorffor cell counting. Determine cell count via Trypan Blue exclusion. Spindown remaining cells, carefully remove supernatant with a pipette (Donot aspirate), and resuspend pellet in 100 microliters of chilled lysisbuffer per 3×10⁶ cells. Incubate cells on ice for 10 minutes then vortexfor 5 seconds. Centrifuge for 1 minute in a microcentrifuge (10,000×g).Transfer supernatant (cytosol extract) to a fresh tube and freeze at−20° C.

Caspase Activity Measurement

[0869] Determine protein concentration by BCA method (Pierce, BCAProtein Assay Kit, #23225). Dilute each supernatant 1:8 in water priorto adding to assay plate. Add all samples and standards in duplicate.After determining the protein concentration of each sample, dilute eachcytosol extract to a concentration of 50 micrograms protein per 50microliters Cell Lysis Buffer (#BY01, Biosource International 1.0mg/ml). Set up assay on a 96 well microtiter U-bottom plate. Useduplicate samples of 50 micrograms protein for caspase assay. Includesamples to be tested without added substrate as a negative control. Add50 microliters of sample per well. Add 50 microliters of 2×ReactionBuffer (#BR01, Biosource International) containing 10 mM DTT to eachsample. Add 5 microliters of the 4 mM substrate (Caspase-3 substrate,#77-900, Biosource International, 200 micromolar final concentration)and incubate at 37° C. for 1.5 hours. Read sample in at 405 nmmicroplate reader. Subtract the no substrate values from the datasamples. Fold-increase in caspase activity can be determined by directcomparison to the level of the uninduced control.

[0870] B. ATF2 Kinase Assay.

[0871] The p38/JNK kinases are members of the MAP Kinase signaltransduction family. Activation of p38 and JNK occurs prior to apoptosisin a number of cell types. To quantitate p38/JNK kinase activity, thephosphorylation of a peptide substrate (ATF2) is measured. Both p38 andJNK, but not other MAP kinases such as ERKs, phosphorylate the ATF2substrate. An enzyme-linked antibody specific for the phosphorylatedATF2 substrate is allowed to bind after reaction of the substrate withextracts from cells treated with controls or TNF-gamma-beta, and theamount of bound antibody is detected spectrophotometrically.

[0872] Confluent BAEC cells were serum-starved in EBM (1 ml per well)for 1 hour. Cells were then treated with TNF-gamma-beta at 10micrograms/ml for 0, 10, 20, 40, 60, 80, 100, 120, 140, 160, or 180minutes. TNFalpha was added to a control well for 20 minutes. After thetreatment, the cells were isolated and assayed for JNK/p38 activity.Cultures were analyzed in duplicate.

[0873] Using the ATF2 assay, TNF-gamma-beta (at 10 micrograms/ml)induced the activation of p38/JNK in a time-dependent manner. Peakactivation (>4-fold over background) occurred 100 minutes after additionof TNF-gamma-beta to BAEC, and the activities persisted for at least thenext hour. TNFalpha control strongly activated p38 and JNK with fasterkinetics (>1100% of negative control at the 20 minute time point).

[0874] The dose-dependancy of TNF-gamma-beta treatment was analyzed bytreatment of the cells with 0, 0.004, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5,5, 10, and 20 micrograms per ml for 80 minutes. Cell lysates were thenisolated and assayed for JNK/p38 activity. Cultures were again analyzedin duplicate. TNF-gamma-beta stimulated JNK/p38 kinase activity in BAECcells in a dose dependent manner. Maximum response (280% of untreatedcontrol) was observed at a TNF-gamma-beta concentration of 20micrograms/ml.

[0875] A detailed ATF2 kinase assay protocol is as follows.

Cell Culture

[0876] BAEC were serum-starved in EBM (Clonetics, 1 ml per well) for 1hour. Cells were then treated with TNF-gamma-beta or TNFa control forindicated times at 37° C. After treatment, cells were rinsed once withice-cold PBS. Cell lists were prepared by adding 0.1 ml (per well of a6-well plate) of lysine buffer (20 mm Tris-Cl [pH7.5], 250 mM NaCl, 0.5%NP-40, 10% Glycerol, 3 mM EDTA, 3 mM EGTA, 0.5 mM sodium orthovanadate,1 mM NaF, 1 mM DTT, 1×Boehringer-Mannheim Complete protease inhibitor)to the cells, and incubating for 2 minutes. Cell debris and nuclei wereremoved by centrifugation. Total protein concentration of the lysateswas determined by BCA assa9 (sigma)

Measurement of ATF2 Phosphorylation

[0877] Coat microlite 2 plate (Dynex) with of a 10 micrograms/mlsolution of GST-ATF2 (residues 19-96) fusion protein (BostonBiologicals), 50 microliters/well. Seal and incubate overnight at roomtemperature (RT). Wash plate once with wash buffer (PBST) (0.05% Tween20, PBS). Block unoccupied sites with 150 microliters/well of blockingbuffer (1.0% Nonfat Dry Milk, prepared in PBS+0.05% Tween 20 [PBST]).Seal and incubate for 60 minutes at RT. Wash plate three times withPBST. Combine 20 microliters/well of kinase buffer (50 mM Hepes [pH7.5], 10 mM MgCl₂, 2.5mM NaF, 0.1 mM sodium orthovanadate, 0.02% BSA[fatty acid-free], 0.5mM DTT, 0.5mM ATP) and 30 microliters/well of celllysate to each well. Seal and incubate for 1.5 hours at RT. Wash platethree times with PBST. Add 50 microliters/well of phospho-specific ATF2antibody (NEB detecting ATF-2 phosphorylated on Thr7 1) that had beendiluted 1:1000. Seal and incubate for 60 minutes at RT. Wash plate threetimes with PBST. Add 50 microliters/well of AMDEX goat anti-rabbitIgG-alkaline phosphatase (Amersham Pharmacia) that had been diluted1:8000. Seal and incubate for 60 minutes at RT. Add 50 microliters/wellof BM chemiluminescent ELISA AP substrate (Roche) to each well. Incubatefor 12 minutes at RT and read on a luminometer at a measurement time of0.1 seconds/well.

[0878] For a reference of the assay, see, Forrer, P., Tamaskovic R., andJaussi, R. (1998). Enzyme-Linked Immunosorbent Assay for Measurement ofJNK, ERK, and p38 Kinase Activities; Biol. Chem. 379(8-9): 1101-1110.

Example 29 Effect of TNF-Gamma on Anti-Angiogenesis in the Cornea

[0879] One of ordinary skill in the art would immediately recognize thatthe following example may be performed essentially as described for anyTNF-gamma protein of the invention (i.e., TNF-gamma-alpha and/orTNF-gamma-beta). The corneal angiogenic assay has been recognizedcommonly as an in vivo assay to evaluate both angiogenic andanti-angiogenic activity of compounds.

[0880] Basic FGF (“bFGF”) has been tested in this corneal angiogenicassay in various research laboratories for a variety of purposes. It hasbeen shown that bFGF can induce significant angiogenesis in corneas(e.g., in rabbits, rats and mice). Based on the previous studies, bFGFwas used in our study of TNF-gamma as an angiogenic factor to induce thegrowth of blood vessels in rat corneas.

[0881] The procedure is relatively common and well known in the art andwas performed essentially as follows.

[0882] Microsurgical implantation in the cornea:

[0883] An incision, 1-1.5 mm in length, is made in the center of thecornea with a microsurgery scalpel blade.

[0884] Starting at the incision, a micropocket is created between thecollagenous layers of the corneal stroma with a Castroviegocyclodialysis spatula. This pocket extends to a point 1-2 mm from thecapillary bed at the corneal-scleral limbus.

[0885] bFGF, which is incorporated 1:1 into non-inflammatory Hydronpolymer (available from Interferon Sciences, New Brunswick, N.J.), isimplanted into the pocket.

[0886] The implanted eyes receive several drops of neosporin ophthalmicantibiotic post-surgery.

[0887] Corneal blood perfusion and harvesting.

[0888] Five or seven days after corneal implantation, corneal bloodperfusion with colloidal carbon is performed. Corneas are thenharvested, fixed, flattened, mounted and photographed for morphology.

[0889] 3. Quantification of angiogenesis.

[0890] An image analysis system (IPLab) is used to quantify the cornealangiogenesis. The corneal surface area, which is covered by the newblood vessels, is quantified and used in our current study. Aftercorneal implantation surgery, TNF-gamma, angiostatin or PBS in a volumeof 25 microliters was injected into the conjunctival region adjacent tothe implantation site. Based on molarities of TNF-gamma and angiostatin,10 micrograms of TNF-gamma or 5 micrograms of angiostatin were givenrespectively four times, once a day, during the first four dayspost-surgery. TNF-gamma and angiostatin were prepared in PBS.

[0891] The experimental grouping was follows: Corneal ImplantsTreatment: PBS bFGF (300 ng) PBS 6 eyes 6 eyes Angiostatin 6 eyes 6 eyesTNF-gamma 6 eyes 6 eyes Total no. eyes: 18 18

[0892] Seven days after surgery, corneas were harvested and processedfor analysis. Angiogenesis was found in the corneas implanted with bFGF,but not in the corneas implanted with pellets containing PBS. Aftermultiple conjunctival injections with PBS, angiostatin or TNF-gamma,there was no obvious vessel growth that was induced by any of thoseinjections in the PBS-implanted corneas. However, conjunctivalinjections with angiostatin and TNF-gamma significantly slowed down theangiogenic process when compared to the conjuctival injections with PBSin the bFGF-implanted corneas. Thus, TNF-gamma inhibited the growth ofblood vessels in corneal angiogenesis assay.

[0893] The following protocol may be used to analyze the effects ofTNF-gamma on bFGF-induced neovascularization in a rat corneal pocketassay in place or in combination with the protocol set forth above.

[0894] bFGF was used to induce neovascularization of the cornea frompreexisting pericorneal limbal vessels in two different versions of theassay. First, corneas were surgically implanted with hydrogel plugscontaining bFGF. TNF-gamma-beta (0.3, 3.0 or 30 μg) or control proteins(angiostatin, 0.15 and 1.5 μg or myeloid progenitor inhibitory factor,5.5 μg) were injected subconjunctivally every day for four days in theconjunctival region 1 mm beyond the pericorneal vasculature in thevicinity of where the hydrogel plug was implanted within the cornea. Ina separate study using an alternate version of this assay, both bFGF andtreatment protein were co-administered on a nitrocellulose filter diskimplanted within the rat corneal stroma. Prior to implantation, filterdisks were treated with phosphate-buffered saline alone, 1.5 μg/μl BSA,1.5 μg/μl TNF-gamma-beta, or 0.05 μg/μl bFGF plus either 1.5 μg/μl BSA,0.15-1.5 μg/μl TNF-gamma-beta, or 3 μg/μl angiostatin. In both assays,the surface area of the angiogenic response was quantitated from digitalimages of treated corneas five days following implantation.

[0895] In these experiments, treatment with 30 μg TNF-gamma-beta reducedthe FGF-induced neovascularization. The maximal decrease in rat cornealneovascularization induced by bFGF is similar for TNF-gamma-beta andangiostatin. In addition, TNF-gamma-beta administered without a stimulusdoes not induce corneal neovascularization. Inhibition of bFGF-inducedneovascularization by TNF-gamma-beta is dose-dependent. In both systems,negative control proteins had no significant effect on bFGF-inducedcorneal neovascularization. Thus, the antiangiogenic activity exhibitedby TNF-gamma-beta in the rat corneal model is not due to non-specificprotein effects.

[0896] These studies indicate that TNF-gamma-beta inhibits bFGF-inducedangiogenesis in a dose-dependent fashion. The maximal degree ofinhibition of angiogenesis produced by TNF-gamma-beta is similar to thatof angiostatin or endostatin.

Example 30 Anti-Angiogenic Activity of TNF-Gamma in Tumors

[0897] Although this example is directed primarily towardsTNF-gamma-beta, one of ordinary skill in the art would immediatelyrecognize that the example may be performed essentially as described forany TNF-gamma protein of the invention (i.e., TNF-gamma-alpha and/orTNF-gamma-beta).

[0898] The in vivo tumor growth in dorsal skin chamber has been avaluable model to directly evaluate angiogenesis and blood flow duringtumor growth. This model provides a direct and continuous means ofnon-invasively quantifying the angiogenesis within growing tumorsthrough transparent windows implanted into the skin.

[0899] Initially, the possible inhibitory effect of TNF-gamma-beta(batch E5) on angiogenesis in the LS 174T colon adenocarcinoma wasanalyzed essentially as follows.

Mouse Preparation

[0900] The surgical procedures were performed in Swiss nude mice. Thesemice were bred and maintained in a defined-flora environment(gnotobiotic; no aerobic flora). For the surgical procedures, animals(20-30 g) were anesthetized with s.c. injection of a cocktail of 90 mgKetamine and 9 mg Xylazine per kg body weight. All surgical procedureswere performed under aseptic conditions in a horizontal laminar flowhood, with all equipment being steam, gas, or chemically sterilized.During surgery, the body temperature of the animals was kept constant bymeans of a heated work surface. All mice were housed individually inmicroisolator cages and all manipulations were done in laminar flowhoods. Buprenorphine (0.1 mg/kg q 12 h) was used as an analgesic for 3days post implantation.

Dorsal Skin Chamber Implantation

[0901] Before implanting chambers, the dorsal skin was prepared withbetadine and positioned such that the chamber sandwiched a double layerof skin that extended above the dorsal surface. One layer of skin wasremoved in a circular area ˜15 mm in diameter. The second layer(consisting of epidermis, fascia, and striated muscle) was positioned onthe frame of the chamber and covered with a sterile, glass coverslip.The titanium and glass chambers were held in place with suture(stainless steel, 4-0) which was threaded through the extended skin andholes along the top of the chamber. Mice were allowed to recover 72hours prior to tumor implantation. The coverslip was carefully removed,followed by the addition of 3 microliters of tumor cell suspension. Thecolon adenocarcinoma, LS174T, was grown in culture to confluence, thentrypsinized and washed twice prior to implantation. A new, sterilecoverslip was placed on the viewing surface following implantation.Observations of tumor growth and angiogenesis were made for 14 daysfollowing the implantation of tumors using intravital microscopy withCCD and SIT cameras. Images were recorded by S-VHS videocasette recorderand direct digital image acquisition.

Experimental Design

[0902] Implanted tumors were allowed to grow in the dorsal chambers for3 days prior to initiation of treatment. Three treatment groups wereused (n=5 mice/group) at 50, 25 and 10 mg/kg, BID, i.p. in PBS. Thecontrol (0 dose group) received 50 mg/ kg BSA in PBS (n=4). Injectionswere made each day for 12 days. Observations of vascular density andtumor growth were made on days 3, 7, 10 and 14 following tumorimplantation. Vascular density was determined by counting individualvessels in adjacent fields across the entire tumor surface and groupmeans were determined for each time point. Tumors were collected formeasurement on day 15 following implantation.

[0903] Suppression of tumor angiogenesis was observed as a result ofTNF-gamma-beta administration in a dose responsive manner. Statisticallysignificant (P<0.05, non-paired t-test) inhibition of new vesselformation could be demonstrated at the 50 mg/kg dose level on days 10and 14. Estimated tumor volumes, based on diameter and thicknessdeterminations, showed decreasing tumor mass with increasing dose ofTNF-gamma-beta. Significant reduction in tumor mass (P<0.05, non-pairedt-test) could be seen at both the 25 and 50 mg/kg dose levels. Detailedobservation of the tumors in situ on day 10 showed the formation ofmicro-hemorrhages at the tumor margin, in contrast to the well formedtumor vessels in the mice treated with the BSA control. The implantedtumor cells in the 25 and 50 mg/kg treatment groups had the appearanceof thin, poorly vascularized discs, while the tumors in the 10 mg/kg andBSA control (0 mg /kg dose group) developed a thickness of up to 3.75mm.

[0904] Thus, these findings indicate that TNF-gamma-beta is a potentinhibitor of angiogenesis in the LS174T tumor.

[0905] In addition to the experimental protocol described above,observations of the establishment of tumor growth and angiogenesis weremade for 17 days following the implantation of tumors using intravitalmicroscopy with CCD cameras. Direct digital images were recorded on aS-VHS videocassette recorder. Implanted tumors were allowed to grow inthe dorsal chambers for 3 days prior to initiation of treatment. Threetreatment groups (n=5 mice/group) received 50, 25 and 10 mg/kgTNF-gamma-beta, BID, via the IP route. The control (0 dose group)received 50 mg/kg BSA in PBS (n=4). Injections were made each day for 12days. Observations of vascular density and tumor growth were made ondays 3, 7, 10 and 14 following tumor implantation. Vascular density wasdetermined by counting individual vessels in adjacent fields across theentire tumor surface and group means were determined for each timepoint. Tumors were collected for measurement following the last day oftreatment. Vascular densities in TNF-gamma-beta-treated mice weresignificantly reduced at the 50 mg/kg dose (IP) on days 7, 10, and 14.

[0906] Following termination of the study, the tumor volumes werecalculated from direct measurement. The data indicate thatTNF-gamma-beta significantly suppressed tumor growth at the 50 and 25mg/kg doses, but not the 10 mg/kg dose (IP). Observation of the tumorsat day 14 indicated disruption of neovascular formation at the peripheryof the implanted tumors at the 50 mg/kg dose. In contrast, tumorstreated with vehicle and BSA showed a typical and well developedvascular structure for the LS174T tumor. These findings suggest thatTNF-gamma-beta may be able to directly interfere with the remodeling ofexisting tumor vasculature.

[0907] Experiments have also been performed to analyze the effect ofTNF-gamma-beta on angiogenesis in an established human adenocarcinomaxenograft. The LS174T colon adenocarcinoma was used to determine ifTNF-gamma-beta could reduce the vascular density in an establishedtumor. Mice were implanted with the dorsal chambers and tumors asdescribed previously. However, the tumors were permitted to grow for 10days prior to initiation of treatment. Mice were then administeredTNF-gamma-beta in three treatment groups (n=5 mice/group) at 50, and 25mg/kg TNF-gamma-beta, BID, IP in PBS. The control (0 dose group)received 50 mg/kg BSA in PBS. Injections were made each day for 7 days.Vascular density was determined by counting individual vessels inadjacent fields across the entire tumor surface and group means weredetermined for each time point.

[0908] Comparison of vessel densities before treatment and following 7days of TNF-gamma-beta administration showed a significant reduction inthe vascular density of the tumor xenografts at the 50 mg/kg dose and asimilar but non-significant trend at the 25 mg/kg dose. Vessel densitydid not significantly decrease in the BSA control tumors. These findingsindicate that the existing vasculature in established tumors can beaffected by TNF-gamma-beta treatment at the same doses that preventearly vessel development in microscopic tumors.

[0909] The effects of TNF-gamma-beta on murine (syngeneic) primary tumorgrowth were also analyzed. The effect of TNF-gamma-beta on the growth ofa murine tumor was performed with the Lewis lung carcinoma (a murinelung adenocarcinoma) in C57BL/6 mice. The syngeneic Lewis lung carcinomatumor model has been utilized by several laboratories in the assessmentof the antitumor properties of a variety of antiangiogenic and otherantitumor agents (Kobayashi et al., 1994; O'Reilly et al., 1994). Toassess the activity of TNF-gamma-beta on Lewis lung carcinoma primarytumor growth, TNF-gamma-beta (10-50 mg/kg) was administered twice dailyfor 14 days by tail vein injection beginning three days followingsubcutaneous inoculation of 1×10⁶ Lewis lung carcinoma cells in thedorsum of male C57BL/6 mice (n=6/group). Primary tumor volumes werecalculated three times per week. Tumor growth was followed out topost-inoculation day 20.

[0910] Twice daily administration of TNF-gamma-beta significantlyinhibited Lewis lung carcinoma primary tumor growth throughout the 14day dosing period. Tumor growth also appears to resume followingwithdrawal of the drug, indicating the reversibility ofTNF-gamma-beta-mediated inhibition. The maximal degree of reduction intumor volume induced by TNF-gamma-beta was approximately 80% relative tothe vehicle control. Since all doses produced a maximal response, theED₅₀ for TNF-gamma-beta inhibition of Lewis lung carcinoma tumor growthin this model is less than 10 mg/kg bid.

[0911] In a subsequent study, the antitumor activity of twice dailydosing and once per day dosing regimen in the Lewis lung carcinoma modelwas examined. 72 hours following tumor inoculation, treatment withTNF-gamma-beta began, with animals receiving 0, 1, 3, or 10 mg/kg iv at250 μl per injection twice per day, or an injection of 0, 3, 10, or 30mg/kg once per day. The injections continued for 14 days, for a total of14 or 28 injections in each animal. When treated twice per day,TNF-gamma-beta treated animals displayed a significant reduction inprimary tumor volume when compared with vehicle-treated controls.However, single daily dosing of TNF-gamma-beta was not sufficient toinhibit tumor growth at any of the concentrations tested. The tumorvolumes of the animals treated twice per day with TNF-gamma-beta did notdiffer significantly from one another. There was no significantdifference apparent in the appearance and general health of animals thatwere treated twice per day with TNF-gamma-beta in any of the dosingconditions. Likewise, there was also no difference seen between thevehicle-treated mice in the once or twice-per-day dosing regimen. Thesestudies indicate that a dose as low as 1 mg/kg, IV bid, is sufficient toproduce inhibition of tumor growth. However, single daily administrationIV is not effective in limiting tumor growth.

Example 31 Endothelial Cell Proliferation Assay (Alamar Blue)

[0912] Alamar blue is an oxidation-reduction indicator that bothfluoresces and changes color in response to chemical reduction of growthmedium resulting from cell growth. The innate metabolic activity ofcells can be measured using Alamar Blue dye. Alamar Blue functions as anelectron acceptor that can be reduced by metabolic intermediates (NADPH,FADH, FMNH and cytochromes). Reduction of Alamar Blue is accompanied bya measurable shift in fluorescence. Alamar Blue does not alter theviability of cells. Alamar Blue methodology has been shown to have equalsensitivity as other cell proliferation assays such as ³H-thymidineincorporation or MTT reduction. Fluorescence measurements are made byexcitation at 530-560 nm and measuring emission at 590 nm. As cells growin culture, innate metabolic activity results in a chemical reduction ofthe immediate surrounding environment. Reduction related to growthcauses the indicator to change from oxidized (non-fluorescent blue) formto reduced (fluorescent red) form and the total signal is proportionalto the total number of cells as well as their metabolic activity.

[0913] Endothelial proliferation is an integral process in tumorangiogenesis. A relevant and customary assay is evaluation of theeffects of anti-angiogenic agents on the suppression of endothelial cellproliferation. TNF-gamma-beta was directly assessed for its growthinhibitory activity on bovine aortic endothelial cells (BAEC) by alamarblue growth assays. Growth of BAEC was induced by treatment of thecultures with 0.5% serum and 1 ng/ml bFGF plus or minus TNF-gamma-beta.Growth was assessed after four days by alamar blue fluorescence. Assaysincluded parallel analysis of TNFalpha inhibition of BAEC growth as apositive control.

[0914] TNF-gamma-beta was shown to suppress the proliferation of BAEC ina concentration-dependent manner, as determined by alamar blue bioassay.Multiple, independently run assays of triplicate cultures gaveequivalent dose curves. The EC₅₀ for these assays was calculated to be5.0+/−0.6 micrograms/ml. Human aortic endothelial cells were also shownto be sensitive to the anti-proliferative activity of TNF-gamma-beta.

[0915] To test the specificity of TNF-gamma-beta actions, Aortic smoothmuscle cells (AoSMC) and normal human dermal fibroblasts (NHDF) wereincubated with TNF-gamma-beta in low serum media, or with 5 ng/ml bFGFor 20 ng/ml PDGF-BB. TNF-gamma-beta at doses equal to those used withBAEC (i.e., 1.25, 5, 10, and 20 micrograms/ml) did not affect the growthof AoSMC or NHDF after 96 hours.

[0916] A detailed alamar blue proliferation assay protocol is asfollows.

[0917] Bovine aortic endothelial cells (BAEC) were plated into 96 wellplates in Clonetics EGM-MV 24 h prior to assay. A standard alamar blueproliferation assay was prepared in serum-free medium (GIBCO HESFM) with1 ng/ml of bFGF added as a source of endothelial cell stimulation.Dilutions of TNF-gamma-beta protein batches were diluted as indicated.SFM without bFGF was used as a non-stimulated control and Angiostatinwas included as a known inhibitory control.

Materials

[0918] Bovine Aortic Endothelial Cells (BAEC), Clonetics Inc.

[0919] Alamar Blue (Biosource Cat# DAL1100)

[0920] EGM-2 MV 10% FBS +Pen/Strep+Glutamine (BAEC Growth medium)

[0921] GIBCO HESFM, 0.5% FBS (sample dilution media)

Method

[0922] BAECs are seeded in growth media at a density of 2000 cells/wellin a 96 well plate and placed at 37° C., 5% CO₂ overnight. After theovernight incubation of the cells, the growth media is removed andreplaced with GIBCO HESFM with 0.5% FBS. The cells are incubated at 37°C., 5% CO₂ overnight. The cells are treated with the appropriatedilutions of TNF-gamma-beta or TNFalpha protein samples (prepared inSFM) in triplicate wells with bFGF at a concentration of 1 ng/ml.Additional plate controls include triplicate wells without bFGF andwells with bFGF and medium alone. Once the cells have been treated withthe samples, the plates are placed back in the 37° C. incubator for anadditional three days. After three days 10 μl of stock alamar bluesolution is added to each well and the plates are placed back in the 37°C. incubator for four hours. The plates are then read at 530 nmexcitation and 590 nm emission using the CytoFluor fluorescence reader.Direct output is recorded in relative fluorescence units.

Analysis

[0923] Fluorescence units from triplicate samples are averaged and themean and SD values are plotted against the log₁₀ of TNF-gamma-betadilution using the Prism software package (GraphPad software, San Diego,Calif.). The EC₅₀ is determined using a 4 parameter fit of the data.Only a fit with r2 values>0.98 are used for EC₅₀ determinations.

[0924] In another embodiment, the above protocol is modified only by asynchronization of the cell cycle of BAECs in the culture to beanalyzed. Synchronization is obtained by supplementing the GIBCO HESFMwith 0.5% FBS (added after the initial overnight incubation detailedabove) with 6 micrograms/ml per well of aphidicolon (Sigma), and thencontinuing with the protocol described above.

Example 32 Endothelial Cell Migration Assay

[0925] Cell migration plays a central role in a wide variety of tissuesduring remodeling processes including angiogenesis. The effect ofTNF-gamma-beta on migration was assessed using in vitro wounding of aconfluent monolayer of BAEC coupled with a computer-assisted system toautomatically collect and analyze the migration data. This model has theadvantage of preserving the special relationship of migrating andnon-migrating cells in the monolayer as well as allowing fast andreliable quantitation.

[0926] A migration index is calculated for each well and represents themean±SD of the cumulative migration distances for treated cells intriplicate wells. A uniform section of the BAEC monolayer was removedwith a plastic probe and cells were cultured in media plus BFGF (10ng/ml) alone, or with 0, 1, 5, 10 or 20 micrograms/ml of TNF-gamma-beta.Image analysis and determination of the migration index was performedafter 24 hours.

[0927] This assay system has established that TNF-gamma-beta inhibitsmigration of BAEC. TNF-gamma-beta inhibited the migration of the BAEC inthe dose-dependant manner. TNF-gamma-beta significantly inhibited BAECmigration at the 10 and 20 micrograms/ml concentration.

[0928] A detailed protocol for an endothelial cell migration assay is asfollows. The following procedures are used for developing asemi-automated system for detecting the migration of the endothelialcells in response to the novel agents.

[0929] Wound a confluent monolayer of the Bovine Aortic EndothelialCells (BAEC) cultured in the 96 well plate. Treat the cells with serafree endothelial cell culture medium containing the agent(s), intriplicate for each concentration, and incubate the cells at 37° C., 5%CO₂ for 24 hours. Fix and stain the cells with 10% formalin and 0.1%crystal violet. Create a digital mask in the NIH Image software bymodeling the actual size and shape of the original wound area. Then usea formula for calculating the actual distance of each cell migrated fromthe either side of the wound edges. Acquire and save the images of thecells around the wound using digital video microscopy and imageprocessing software. Cover the image of one field a time with a digitalmask to block the cells distributed in the non-wound area and exposeonly the migrating cells. Capture and counts the cells in the wound areaand then measure the raw distance of each cell alone the X-axis. Use theformula to determine the actual distance migrated from either edge ofthe wounded area of the monolayer. Total the distances of the all cellsfor each group as migration index (MI), which represents the net effectof an agent on the in vitro migration of the endothelial cells.

Example 33 TNF-Gamma Binding Assays Preparation of RadiolabeledTNF-Gamma-Beta

[0930] Radio-iodination of TNF-gamma-beta was performed using theIodobead method. Briefly, one Iodobead (Pierce) per reaction waspre-washed with PBS and added to 1 mCi of NaI¹²⁵ in 80 microliters ofPBS pH 6.5. The reaction was allowed to proceed for 5 minutes and then10 micrograms of TNF-gamma-beta was added and incubated for 5 minutes atroom temperature. Iodinated protein was separated from unboundradioactivity using a G-25 Sephadex quick spin column previouslyequilibrated with PBS containing 0.1% BSA. Protein concentration andspecific radioactivity of I¹²⁵-Vasolysin were determined by TCAprecipitation of pre-column and post-column samples. The specificactivity of I¹²⁵-Vasolysin used in the experiment was 15.2 microcuriesper microgram.

Competitive Binding Assay to Determine Specific Binding

[0931] BAEC, HAEC and NHDF cells were plated (2×10⁵ cells/well) in 24well plate overnight. The binding assay was performed in 500 microlitersof binding buffer (Ham's F containing 0.5% BSA and 0.1% sodium azide)containing 0.3 nM I¹²⁵-TNF-gamma-beta in the absence or presence of100-fold excess of unlabeled TNF-gamma-beta. Binding to cells wasperformed in triplicates in a 96 well plate using 1×10⁶ cells in 100microliters of binding buffer under similar conditions used for othercell types with 0.3 nM ¹²⁵I-TNF-gamma-beta. The binding reaction wascarried out at room temperature for 2 hr. Cell bound I¹²⁵-TNF-gamma-betawas separated from unbound material by centrifugation through 200microliters of 1.5 dibutylphthlate/1.0 bis (2-ethyl-hexyl) phthalate oilmixture in a polyethylene microfuge tubes (Bio-Rad) for 20 sec at 12,000RPM. The microfuge tubes were then frozen quickly in liquid nitrogen andthe bottom tip of the tubes was cut off using a tube cutter.Radioactivity in the bottom containing the cell pellet (bound fraction)and the top (unbound fraction) of the tubes were counted by using agamma counter.

[0932] The cells were then washed three times with PBS containing 0.1%BSA and lysed with 1% NP40 solution and counted using a gamma counter.

[0933] To determine affinity (Kd) of TNF-gamma-beta binding to cells,binding assay was performed with 0.3 nM I¹²⁵-TNF-gamma-beta in presenceof increasing concentrations of unlabeled TNF-gamma-beta (0.01 to 639nM). The data was analyzed by Prizm software (GraphPad Software, SanDiego, Calif.) to determine dissociation constant (Kd) and number ofbinding sites.

Example 34 Generation and Characterization of anti-TNF-gamma-betaAntibodies

[0934] Balb/C mice were immunized with TNF-gamma-beta polypeptide (aminoacid residues 72-251 of SEQ ID NO:20 according to the followingschedule: Day Dose/mouse Route Vehicle  1 50 micrograms Sub-cutaneousComplete Freund's Adjuvant 13 50 micrograms Sub-cutaneous IncompleteFreund's Adjuvant 27 50 micrograms Sub-cutaneous Incomplete Freund'sAdjuvant 38 10 micrograms Intra-peritoneal PBS 59 10 microgramsIntra-peritoneal PBS 97 10 micrograms Intra-peritoneal PBS

[0935] After the final immunization, hybridomas were generated accordingto standard protocols. Hybridomas were initially screened by ELISA fortheir ability to bind TNF-gamma-beta (amino acid residues 72-251 of SEQID NO:20) by ELISA which identified eighteen positive hybridomas: 03C06,04H08, 06C03, 06D09, 06F03, 08D06, 12D08, 12F11, 14A03, 15B03, 15E09,16B05, 16H02, 17A03, 17D07, 18G08, 20B01 and 20C05.

Characterization of Murine Monoclonal Anti-TNF-gamma-beta Antibodies

[0936] TNF-gamma-beta treatment induces production of secreted alkalinephosphatase in TF-1/SRE reporter cells. Additionally TNF-gamma-betatreatment results in caspase activation in TF-1 cells. The ability ofthe murine monoclonal antibodies to neutralize these TNF-gamma-betamediated activities were investigated.

SEAP Assay

[0937] The ability of TNF-gamma-beta to generate a signal that activatesgenes under the regulation of Signal Response Elements (SREs) wasexamined using TF-1 cell line transfected with an SRE/Secreted AlkalinePhosphatase (SEAP) reporter plasmid. Briefly, a poly-D-lysine coated96-well plate is seeded with TF-1/SRE-SEAP cells (in RPMI+0.2% Fetalbovine serum) at 75,000 cells per well. Cells were incubated overnightand the media was aspirated the next morning and replaced with media(RPMI +0.2% fetal bovine serum) containing TNF-gamma-beta. Again cellswere incubated overnight. After overnight incubation, conditioned mediawere collected and SEAP activity was determined using the SEAP ReporterGene Assay available from Roche Molecular Biochemicals (Indianapolis,Ind.) according to the manufacturer's directions. Briefly, Conditionedmedia were diluted 1:4 into dilution buffer. Samples were incubated at65C for 30 minutes to eliminate contaminating AP activity usuallypresent in culture medium. 25 microliters of the heat-inactivatedsamples were mixed with equal volume of inactivation buffer (containinga mixture of differential alkaline phosphatase inhibitors). Following a5-minute incubation at room temperature, 50 uL of alkaline phosphatasesubstrate (CSPD) was added to each well. Chemiluminescence signal wasread 10-15 minutes later using a luminometer. TNF-gamma-beta inducesSEAP production in a dose dependent fashion.

[0938] Antibodies generated against TNF-gamma-beta were tested for theability to inhibit the TNF-gamma-beta induced SEAP production inTF-1/SRE_SEAP reporter cells. Briefly, 24 micrograms/mL of each antibody(50×molar excess) was mixed with either 200 ng/mL of TNF-gamma-beta inmedium (RPMI+0.2% FBS) or in medium alone (RPMI+0.2% FBS) in a totalvolume of 150 microliters. These solutions were then incubated for 1hour at room temperature. Fifty microliters of the media containingTNF-gamma-beta+antibody or antibody alone solution was added to the TF-1cells which were then incubated overnight. After the overnightincubation, the SEAP assay was performed as described above. Using thisassay, monoclonal antibodies 12D08, 14A03, 15E09, and 16H02 wereidentified as potent TNF-gamma-beta neutralizing antibodies.

Caspase Assay

[0939] The ability of TNF-gamma-beta to induce caspase activity in TF-1cells was analyzed using a Homogeneous Fluorimetric Caspases Assayavailable from Roche Molecular Biochemicals (Indianapolis, Ind.)according to the manufacturer's directions. Briefly, cells growing inmicrotiter plates are induced to undergo apoptosis, causing anactivation of caspase activities. Equal volume of a caspase substrate(Asp-Glu-Val-Asp-Rhodamine 110, or DEVD-R1 10) solution is then addedand incubated for at least 1 hour. During this incubation, cells arebeing lysed and free RI 10 is released from the substrate. The level offree R110 is determined fluorimetrically, using a fluorescence readerwith excitation filter 470-500 nm and emission filter 500-560 nm.

[0940] A black 96-well plate with a clear bottom is seeded with 75,000TF-1 cells in RPMI containing 1% fetal bovine serum andmicrograms/milliliter cyclohexamide. An equal volume of 2×TNF-gamma-betais then added to the wells and incubated for 5 hours prior to performingthe caspase assay. Following the manufacturer's directions, an equalvolume of 1×substrate solution containing 50 micromolar DEVD-R110diluted in incubation buffer is added to each well. The 96-well platesare then incubated for 2 hours after which the plate is read in afluorescence reader with an excitation filter at 485 nm and an emissionfilter at 535 nm. TNF-gamma-beta induces caspase production in a dosedependent fashion.

[0941] Antibodies generated against TNF-gamma-beta were tested for theability to inhibit the TNF-gamma-beta induced caspase activation.Briefly, 24 micrograms/mL of each antibody (100×molar excess) was mixedwith either 100 ng/mL of TNF-gamma-beta in medium (RPMI+1% FBS+20micrograms/mL cyclohexamide) or in medium alone (RPMI+1% FBS+20micrograms/mL cyclohexamide) in a total volume of 150 microliters. Thesesolutions were then incubated for 1 hour at room temperature. The mediacontaining TNF-gamma-beta+antibody or antibody alone solution were thenadded to the TF-1 cells and the caspase assay was performed as describedabove. Using this assay, monoclonal antibodies 12D08, 14A03, 15E09, and16H02 were identified as potent TNF-gamma-beta neutralizing antibodies.

Example 35 TR6 and DR3 Interact with TNF-Gamma-beta

[0942] The premyeloid cell line TF-1 was stably transfected withSRE/SEAP (Signal Response Element/Secreted Alkaline Phosphatase)reporter plasmid that responds to the SRE signal transduction pathway.The TF1/SRE reporter cells were treated with TNF-gamma-beta at 200 ng/mLand showed activation response as recorded by the SEAP activity. Thisactivity can be neutralized by TR6.fc fusion protein in a dose dependentmanner. The TR6.Fc by itself, in contrast, showed no activity on theTF1/SRE reporter cells. The results demonstrate that 1) TF-1 is a targetcell for TNF-gamma-beta ligand activity. 2) TR6 (InternationalPublication Numbers WO98/30694 and WO00/52028) interacts withTNF-gamma-beta and inhibits its activity on TF-1 cells. TR6 has twosplice forms, alpha and beta; both splice forms have been shown tointeract with TNF-gamma-beta.

[0943] Similarly, the interaction of DR3 (International PublicationNumbers WO97/33904 and WO/0064465) and TNF-gamma-beta can bedemonstrated using TF-1/SRE reporter cells. The results indicate thatDR3.fc interacts with TNF-gamma-beta, either by competing naturallyexpressed DR3 on TF-1 cells or forming inactive TNF-gamma-beta/DR3.fccomplex, or both.

[0944] At least three additional pieces of evidence demonstrate aninteraction between TNF-gamma-beta and DR3 and TR6. First, both TR6.Fcand DR3.Fc are able to inhibit TNF-gamma-beta activation of NFkB in 293Tcells, whereas in the same experiment, TNFRI.Fc was not able to inhibitTNF-gamma-beta activation of NFkB in 293T cells. Secondly, both TR6.Fcand DR3.Fc can be used to immunoprecipitate TNF-gamma-beta. Thirdly,TR6.Fc proteins can be detected by FACS analysis to specifically bindcells transfected with TNF-gamma-beta.

Example 36 T Cell Proliferation and IFN-gamma ELISA T Cell ProliferationAssay

[0945] The assay is performed as follows. PBMCs are purified from singledonor whole blood by centrifugation through a histopaque gradient. PBMCsare cultured overnight in 10% RPMI and the following day non-adherentcells are collected and used for the proliferation assay. 96-well platesare pre-coated with either anti-CD3 or anti-CD3 and anti-CD28 andincubated overnight at 4 C. Plates are washed twice with PBS before use.TNF-gamma-beta protein at desired concentrations in 10% RPMI is added tothe 2×10⁴ cells/well in a final volume of 200 ul. 10 ng/ml recombinanthuman IL2 was used as a positive control. After 24 hours culture,samples are pulsed with 1 uCi/well 3H-thymidine. 26 hours after pulsing,cells are harvested and counted for 3H-thymidine.

IFNgamma ELISA

[0946] The assay is performed as follows. Twenty-four well plates arecoated with either 300 ng/ml or 600 ng/ml anti-CD3 and 5 ug/ml anti-CD28(Pharmingen, San Diego, Calif.) in a final volume of 500 ul andincubated overnight at 4C. Plates are washed twice with PBS before use.PBMC are isolated by Ficoll (LSM, ICN Biotechnologies, Aurora, Ohio)gradient centrifugation from human peripheral blood, and are culturedovernight in 10% FCS(Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI(Gibco BRL, Gaithersburg, Md.). The following day, the non adherentcells are collected, washed and used in the costimulation assay. Theassay is performed in the pre-coated twenty-four well plate using 1×10⁵cells/well in a final volume of 900 ul. TNF-gamma-beta protein is addedto the cultures. Recombinant human IL-2 (purchased from R & D Systems,Minneapolis, Minn.) at a final concentration of 10 ng/ml was used as apositive control. Controls and unknown samples are tested in duplicate.Supernatant samples (250 ul) are collected 2 days and 5 days after thebeginning of the assay. The level of IFN gamma and IL-2 in culturesupernatants is then measured by ELISA.

Results

[0947] TNF-gamma-beta treatment of PBMCs results in proliferation of Tcells and a significant increase in IFN-gamma production compared tocontrols.

Example 37 TNF-gamma-beta Exacerbates an In-vivo MLR Reaction

[0948] Acute graft-versus-host disease (aGVHD) is a major complicationof allogeneic bone marrow transplantation (BMT), which is associatedwith a prolonged immune deficiency leading to life-threateninginfections. The immunopathophysiology of GVHD is complex, and isconsidered to involve two phases. In the inductive phase, donor Tlymphocytes recognize antigen expressed on recipient tissues resultingin alloactivation and proliferation of the allogeneic donor T cells. Inthe effector phase, inflammatory reactions may develop in specific hosttarget tissues such as spleen, liver, intestine and skin that arecharacterized by mononuclear cell infiltration and histopathologicaldamage. Some evidence suggests that GVHD-associated lymphoid hypoplasiaand B cell dysfunction is dependent upon donor T cell-mediated Fasligand function, but not perforin function: Parent-into-Fl mice is arepresentative model of an acute form of GVHD, that is caused bytransfusion of C57BL/6 splenic T cells into (BALB/c×C57BL/6) F1 mice(CB6F1), and resulted in tissue damage of lymphoid, livergastrointestinal tract or skin, and finally death. TNF-gamma-beta, aligand of TNF superfamily, seems to be a T cell costimulator resultingin significant elevation of pro-inflammatory cytokine (INF-g and GM-CSF)secretion. In order to test the effect of TNF-gamma on T cell mediatedalloactivation we co-administered TNF-gamma-beta protein along withC57BL/6 splenic T cells into CB6F1 mice and measured the course of thealloreaction via spleen weight and cytokine production among otherparameters.

[0949] Briefly, CB6F1 mice were injected with 1.5×108 pooled spleencells of C57BL/6 mice administered intravenously on day 0.TNF-gamma-beta, TL5 (a.k.a. AIM-II, another TNF family ligand used hereas a control, or buffer was given intravenously at 3mg/kg/day for 5 days(day 0 through day 4). All mice were sacrificed on day 5 for the purposeof collecting the spleen for weighting, and obtaining cells for cellproliferation and cytokine (IL-2, INF-gamma, GM-CSF, IL-12) productionassays, as well as for FACS analysis.

[0950] Five days after parent splenocyte transfer, the alloactivationwas observed in CB6F1 mice measured by splenomegaly, spontaneoussplenocyte proliferation and cytokine production (buffer group vs.normal control). Treatment with TNF-gamma beta at 3 mg/kg/day, i.v. for5 days resulted in a further significant enhancement of spleen weight(p=0.001 using ANOVA/T-test), the spontaneous splenocyte proliferationand cytokine (GM-CSF, INF-gamma) production when compared with the sameparameters of buffer-treated control group; whereas TL5, showed nostatistically significant difference from buffer group. In addition,neither TNF-gamma-beta nor TL5 show any significant effect on IL-12 andIL-2 production comparing with buffer group. This result suggests thathuman TNF-gamma is effective in vivo for modulating T cell-mediatedimmune response, and its effect on cytokine production may be selective.

[0951] Administration of TNF-gamma-beta protein to allografted miceexacerbates acute graft vs. host disease as measured by early sign ofsplenomegaly, spontaneous splenocyte proliferation and proinflammatorycytokine production suggesting that TNF-gamma-beta may play a pathogenicrole in GVHD. Thus, antagonists of TNF-gamma-beta (e.g., neutralizingantibody against TNF-gamma or soluble DR3 or TR6 proteins such as Fc oralbumin fusion proteins) might have therapeutic potential in treatingpatients with this and other T cell-mediated inflammatory processes anddiseases, including, but not limited to, systemic lupus erythematosus,multiple sclerosis, arthritis, and delayed-type hypersensitivityreactions.

[0952] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

[0953] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

[0954] Further, the Sequence Listing submitted herewith in both computerand paper forms are hereby incorporated by reference in theirentireties. Additionally the specification and Sequence Listing of thefollowing U.S. applications are herein incorporated by reference intheir entirety: U.S. Provisional Application Serial Nos.: 60/074,047,filed Feb. 9, 1998, 60/131,963, filed on Apr. 30, 1999; 60/132,227,filed May 3, 1999; and 60/134,067, filed May 13, 1999, 60/180,908, filedFeb. 8, 2000, 60/216,879, filed Jul. 7, 2000, and 60/278,449 filed Mar.26, 2001; U.S. nonprovisional application Ser. Nos.: 08/461,246, filedJun. 5, 1995, 09/005,020, filed Jan. 9, 1998, 60/074,047, filed Feb. 9,1998, 09/131,237, filed Aug. 7, 1998, 09/246,129, filed Feb. 8, 1999,09/559,290, filed Apr. 27, 2000; and 09/560,921 filed 4/28/2000 each ofwhich is hereby incorporated by reference in its entirety; and PCTApplication Serial Nos. PCT/US94/12880, filed Nov. 7, 1994,PCT/US99/02722, filed Feb. 8, 1999, PCT/US00/11689; each of which ishereby incorporated by reference in its entirety.

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acctcacacc tagagttcct atacctctgagactccagag gaaagaacaa 120 gacagtgcag aaggatatgt tagaacccac tgaaaacctagaaggttgaa aaggaagcat 180 accctcctga cctataagaa aattttcagt ctgcagggggatatccttgt ggcccaagac 240 attggtgtta tcatttgact aagaggaaat tatttgtggtgagctctgag tgaggattag 300 gaccagggag atgccaagtt tctatcactt acctcatgcctgtaagacaa gtgttttgtt 360 ccaattgatg aatggggaga aaacagttca gccaatcacttatgggcaca gaatggaatt 420 tgaagggtct ggtgcctgcc cttgtcatac gtaaacaagagaggcatcga tgagttttat 480 ctgagtcatt tgggaaagga taattcttgc accaagccattttcctaaac acagaagaat 540 agggggattc cttaaccttc attgttctcc aggatcataggtctcaggat aaattaaaaa 600 ttttcaggtc agaccactca gtctcagaaa ggcaaagtaatttgccccag gtcactagtc 660 caagatgtta ttctctttga acaaatgtgt atgtccagtcacatattctt cattcattcc 720 tccccaaagc agtttttagc tgttaggtat attcgatcactttagtctat tttgaaaatg 780 at atg aga cgc ttt tta agc aaa gtc tac agt ttccca atg aga aaa 827 Met Arg Arg Phe Leu Ser Lys Val Tyr Ser Phe Pro MetArg Lys -25 -20 -15 tta atc ctc ttt ctt gtc ttt cca gtt gtg aga caa actccc aca cag 875 Leu Ile Leu Phe Leu Val Phe Pro Val Val Arg Gln Thr ProThr Gln -10 -5 -1 1 cac ttt aaa aat cag ttc cca gct ctg cac tgg gaa catgaa cta ggc 923 His Phe Lys Asn Gln Phe Pro Ala Leu His Trp Glu His GluLeu Gly 5 10 15 20 ctg gcc ttc acc aag aac cga atg aac tat acc aac aaattc ctg ctg 971 Leu Ala Phe Thr Lys Asn Arg Met Asn Tyr Thr Asn Lys PheLeu Leu 25 30 35 atc cca gag tcg gga gac tac ttc att tac tcc cag gtc acattc cgt 1019 Ile Pro Glu Ser Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr PheArg 40 45 50 ggg atg acc tct gag tgc agt gaa atc aga caa gca ggc cga ccaaac 1067 Gly Met Thr Ser Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn55 60 65 aag cca gac tcc atc act gtg gtc atc acc aag gta aca gac agc tac1115 Lys Pro Asp Ser Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr 7075 80 cct gag cca acc cag ctc ctc atg ggg acc aag tct gta tgc gaa gta1163 Pro Glu Pro Thr Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val 8590 95 100 ggt agc aac tgg ttc cag ccc atc tac ctc gga gcc atg ttc tccttg 1211 Gly Ser Asn Trp Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu105 110 115 caa gaa ggg gac aag cta atg gtg aac gtc agt gac atc tct ttggtg 1259 Gln Glu Gly Asp Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val120 125 130 gat tac aca aaa gaa gat aaa acc ttc ttt gga gcc ttc tta cta1304 Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 135 140145 taggaggaga gcaaatatca ttatatgaaa gtcctctgcc accgagttcc taattttctt1364 tgttcaaatg taattataac caggggtttt cttggggccg ggagtagggg gcattccaca1424 gggacaacgg tttagctatg aaatttgggg ccaaaatttc acacttcatg tgccttactg1484 atgagagtac taactggaaa aaggctgaag agagcaaata tattattaag atgggttgga1544 ggattggcga gtttctaaat attaagacac tgatcactaa atgaatggat gatctactcg1604 ggtcaggatt gaaagagaaa tatttcaaca cctccctgct atacaatggt caccagtggt1664 ccagttattg ttcaatttga tcataaattt gcttcaattc aggagctttg aaggaagtcc1724 aaggaaagct ctagaaaaca gtataaactt tcagaggcaa aatccttcac caatttttcc1784 acatactttc atgccttgcc taaaaaaaat gaaaagagag ttggtatgtc tcatgaatgt1844 tcacacagaa ggagttggtt ttcatgtcat ctacagcata tgagaaaagc tacctttctt1904 ttgattatgt acacagatat ctaaataagg aagtttgagt ttcacatgta tatcccaaat1964 acaacagttg cttgtattca gtagagtttt cttgcccacc tattttgtgc tgggttctac2024 cttaacccag aagacactat gaaaaacaag acagactcca ctcaaaattt atatgaacac2084 cactagatac ttcctgatca aacatcagtc aacatactct aaagaataac tccaagtctt2144 ggccaggcgc agtggctcac acctgtaatc ccaacacttt gggaggccaa ggtgggtgga2204 tcatctaagg ccgggagttc aagaccagcc tgaccaacgt ggagaaaccc catctctact2264 naaaatacna aattagccgg gcgtggtagc gcatggctgt aancctggct actcaggagg2324 ccgaggcaga anaattnctt gaactgggga ggcagaggtt gcggtgagcc cagancgcgc2384 cattgcactc cagcctgggt aacaagagca aaactctgtc caaaaaaaaa aaaaaaaa2442 <210> SEQ ID NO 2 <211> LENGTH: 174 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 2 Met Arg Arg Phe Leu Ser Lys Val Tyr SerPhe Pro Met Arg Lys Leu -25 -20 -15 Ile Leu Phe Leu Val Phe Pro Val ValArg Gln Thr Pro Thr Gln His -10 -5 -1 1 5 Phe Lys Asn Gln Phe Pro AlaLeu His Trp Glu His Glu Leu Gly Leu 10 15 20 Ala Phe Thr Lys Asn Arg MetAsn Tyr Thr Asn Lys Phe Leu Leu Ile 25 30 35 Pro Glu Ser Gly Asp Tyr PheIle Tyr Ser Gln Val Thr Phe Arg Gly 40 45 50 Met Thr Ser Glu Cys Ser GluIle Arg Gln Ala Gly Arg Pro Asn Lys 55 60 65 Pro Asp Ser Ile Thr Val ValIle Thr Lys Val Thr Asp Ser Tyr Pro 70 75 80 85 Glu Pro Thr Gln Leu LeuMet Gly Thr Lys Ser Val Cys Glu Val Gly 90 95 100 Ser Asn Trp Phe GlnPro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln 105 110 115 Glu Gly Asp LysLeu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp 120 125 130 Tyr Thr LysGlu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 135 140 145 <210> SEQ ID NO3 <211> LENGTH: 233 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 3 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu GluAla 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg CysLeu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr ThrLeu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu GluSer Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val ArgSer Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val ValAla Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg AlaAsn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln LeuVal Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val LeuPhe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr HisThr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val AsnLeu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro GluGly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly GlyVal Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile AsnArg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr PheGly Ile Ile Ala Leu 225 230 <210> SEQ ID NO 4 <211> LENGTH: 205 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Met Thr Pro ProGlu Arg Leu Phe Leu Pro Arg Val Cys Gly Thr Thr 1 5 10 15 Leu His LeuLeu Leu Leu Gly Leu Leu Leu Val Leu Leu Pro Gly Ala 20 25 30 Gln Gly LeuPro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala 35 40 45 Arg Gln HisPro Lys Met His Leu Ala His Ser Thr Leu Lys Pro Ala 50 55 60 Ala His LeuIle Gly Asp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg 65 70 75 80 Ala AsnThr Asp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn 85 90 95 Asn SerLeu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln 100 105 110 ValVal Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Pro Ser Ser Pro 115 120 125Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln Tyr Pro Phe 130 135140 His Val Pro Leu Leu Ser Ser Gln Lys Met Val Tyr Pro Gly Leu Gln 145150 155 160 Glu Pro Trp Leu His Ser Met Tyr His Gly Ala Ala Phe Gln LeuThr 165 170 175 Gln Gly Asp Gln Leu Ser Thr His Thr Asp Gly Ile Pro HisLeu Val 180 185 190 Leu Ser Pro Ser Thr Val Phe Phe Gly Ala Phe Ala Leu195 200 205 <210> SEQ ID NO 5 <211> LENGTH: 244 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 5 Met Gly Ala Leu Gly Leu Glu GlyArg Gly Gly Arg Leu Gln Gly Arg 1 5 10 15 Gly Ser Leu Leu Leu Ala ValAla Gly Ala Thr Ser Leu Val Thr Leu 20 25 30 Leu Leu Ala Val Pro Ile ThrVal Leu Ala Val Leu Ala Leu Val Pro 35 40 45 Gln Asp Gln Gly Gly Leu ValThr Glu Thr Ala Asp Pro Gly Ala Gln 50 55 60 Ala Gln Gln Gly Leu Gly PheGln Lys Leu Pro Glu Glu Glu Pro Glu 65 70 75 80 Thr Asp Leu Ser Pro GlyLeu Pro Ala Ala His Leu Ile Gly Ala Pro 85 90 95 Leu Lys Gly Gln Gly LeuGly Trp Glu Thr Thr Lys Glu Gln Ala Phe 100 105 110 Leu Thr Ser Gly ThrGln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro 115 120 125 Gln Asp Gly LeuTyr Tyr Leu Tyr Cys Leu Val Gly Tyr Arg Gly Arg 130 135 140 Ala Pro ProGly Gly Gly Asp Pro Gln Gly Arg Ser Val Thr Leu Arg 145 150 155 160 SerSer Leu Tyr Arg Ala Gly Gly Ala Tyr Gly Pro Gly Thr Pro Glu 165 170 175Leu Leu Leu Glu Gly Ala Glu Thr Val Thr Pro Val Leu Asp Pro Ala 180 185190 Arg Arg Gln Gly Tyr Gly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly 195200 205 Gly Leu Val Gln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser210 215 220 His Pro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe GlyAla 225 230 235 240 Val Met Val Gly <210> SEQ ID NO 6 <211> LENGTH: 278<212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE: 6 MetGln Gln Pro Val Asn Tyr Pro Cys Pro Gln Ile Tyr Trp Val Asp 1 5 10 15Ser Ser Ala Thr Ser Pro Trp Ala Pro Pro Gly Ser Val Phe Ser Cys 20 25 30Pro Ser Ser Gly Pro Arg Gly Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45Pro Pro Pro Pro Ser Pro Leu Pro Pro Pro Ser Gln Pro Pro Pro Leu 50 55 60Pro Pro Leu Ser Pro Leu Lys Lys Lys Asp Asn Ile Glu Leu Trp Leu 65 70 7580 Pro Val Ile Phe Phe Met Val Leu Val Ala Leu Val Gly Met Gly Leu 85 9095 Gly Met Tyr Gln Leu Phe His Leu Gln Lys Glu Leu Ala Glu Leu Arg 100105 110 Glu Phe Thr Asn His Ser Leu Arg Val Ser Ser Phe Glu Lys Gln Ile115 120 125 Ala Asn Pro Ser Thr Pro Ser Glu Thr Lys Lys Pro Arg Ser ValAla 130 135 140 His Leu Thr Gly Asn Pro Arg Ser Arg Ser Ile Pro Leu GluTrp Glu 145 150 155 160 Asp Thr Tyr Gly Thr Ala Leu Ile Ser Gly Val LysTyr Lys Lys Gly 165 170 175 Gly Leu Val Ile Asn Glu Ala Gly Leu Tyr PheVal Tyr Ser Lys Val 180 185 190 Tyr Phe Arg Gly Gln Ser Cys Asn Ser GlnPro Leu Ser His Lys Val 195 200 205 Tyr Met Arg Asn Phe Lys Tyr Pro GlyAsp Leu Val Leu Met Glu Glu 210 215 220 Lys Lys Leu Asn Tyr Cys Thr ThrGly Gln Ile Trp Ala His Ser Ser 225 230 235 240 Tyr Leu Gly Ala Val PheAsn Leu Thr Val Ala Asp His Leu Tyr Val 245 250 255 Asn Ile Ser Gln LeuSer Leu Ile Asn Phe Glu Glu Ser Lys Thr Phe 260 265 270 Phe Gly Leu TyrLys Leu 275 <210> SEQ ID NO 7 <211> LENGTH: 235 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 7 Met Ser Thr Glu Ser Met Ile ArgAsp Val Glu Leu Ala Glu Gly Pro 1 5 10 15 Leu Pro Lys Lys Ala Gly GlyPro Gln Gly Ser Lys Arg Cys Leu Cys 20 25 30 Leu Ser Leu Phe Ser Phe LeuLeu Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Arg ValIle Gly Pro Gln Glu Glu Glu Gln Ser 50 55 60 Pro Asn Asn Leu His Leu ValAsn Pro Val Ala Gln Met Val Thr Leu 65 70 75 80 Arg Ser Ala Ser Arg AlaLeu Ser Asp Lys Pro Leu Ala His Val Val 85 90 95 Ala Asn Pro Gln Val GluGly Gln Leu Gln Trp Leu Ser Gln Arg Ala 100 105 110 Asn Ala Leu Leu AlaAsn Gly Met Lys Leu Thr Asp Asn Gln Leu Val 115 120 125 Val Pro Ala AspGly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Ser 130 135 140 Gly Gln GlyCys Arg Ser Tyr Val Leu Leu Thr His Thr Val Ser Arg 145 150 155 160 PheAla Val Ser Tyr Pro Asn Lys Val Asn Leu Leu Ser Ala Ile Lys 165 170 175Ser Pro Cys His Arg Glu Thr Pro Glu Glu Ala Glu Pro Met Ala Trp 180 185190 Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp 195200 205 Arg Leu Ser Thr Glu Val Asn Gln Pro Glu Tyr Leu Asp Leu Ala Glu210 215 220 Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230 235<210> SEQ ID NO 8 <211> LENGTH: 434 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (15)<223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (19) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (133) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (388) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (424) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 8tctacacaag gtacngacng ctaccctgag ccaacccagc tcctcatggg gaccaagtct 60gtatgcgaag taggtagcaa ctggttccag cccatctacc tcggagccat gttctccttg 120caagaagggg acnagctaat ggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 180gaagataaaa ccttctttgg agccttctta ctataggagg agagcaaata tcattatatg 240aaagtcctct gccaccgagt tcctaatttt ctttgttcaa atgtaattat aaccaggggt 300tttcttgggg ccgggagtag ggggcattcc cacagggaca acggtttagc tatgaaattt 360ggggggccca aaatttcaca acttcatngt tgcccttact tgatgagaag tacttaactt 420gganaaaagg cttg 434 <210> SEQ ID NO 9 <211> LENGTH: 493 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (288) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (296) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (309) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (314) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (340) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (343) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (348) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (369) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (385) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (410) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (417) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (423) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (431) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (434) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (437) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (444) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (459) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (486) <223> OTHERINFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 9 aattcggcagagaaattcca tactatcacc agttggccaa ctttccaagt ctagtgcaga 60 aatccaaggcacctcacacc tagagttcct atacctctga gactccagag gaaagaacaa 120 gacagtgcagaaggatatgt tagaacccac tgaaaaccta gaaggttaaa aaggaagcat 180 accctcctgacctataagaa aattttcagt ctgcaggggg atatccttgt ggcccaagac 240 attggtgttatcatttgact aagaggaaat tatttgtggt gagctccnag tgaggnttag 300 ggaccaggnggtgnccaagt ttctatcact tacctcatgn ctntaagnca agtgttttgt 360 tcccattgntgatggggtta aaacnttcag ccatcacttt tggggcaagn atggggnttt 420 gangggttggngcnggnctt gtcntcgtaa acagggggnt tggtgggttt ttctgggtcc 480 ttgggnaggactt 493 <210> SEQ ID NO 10 <211> LENGTH: 380 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (53)..(54) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (258) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (316) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (324) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (346) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (367) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (378) <223> OTHER INFORMATION: n equals to a, t, g, or c<400> SEQUENCE: 10 ggcagaggtt caatttgatc ataaatttgc ttcaattcaggagctttgaa ggnngtccaa 60 ggaaagctct agaaaacagt ataaactttc agaggcaaaatccttcacca atttttccac 120 atactttcat gccttgccta aaaaaaatga aaagagagttggtatgtctc atggaatgtt 180 cacacagaag gagttggttt tcatgtcatc tacagcatatgagaaaagct acctttcttt 240 tgattatgta cacaggtntc taaataagga agtatgagtttcacatgtat attcaaaaat 300 acaacagttg cttgtnttca gttngggttt ttcttggcccacccantttt ggtgctgggg 360 gttctanctt taaccccnga 380 <210> SEQ ID NO 11<211> LENGTH: 458 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (9) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (12) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (119) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (303) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (311) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (387) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (409) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (425) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (427) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (453) <223> OTHER INFORMATION: n equals to a, t, g, or c<400> SEQUENCE: 11 ggcacagcng gnagtagggg gcattccaca gggacaacggtttagctatg aaatttgggg 60 cccaaaattt cacacttcat gtgccttact gatgagagtactaactggaa aaaggctgna 120 agagagcaaa tatattatta agatgggttg gaggattggcgagtttctaa atattaagac 180 actggatcac tgaaatgaat ggatgatcta ctcgggtccaggattgaaag agaaatattt 240 caacaccttc ctgctataca atggtcacca gtggtccagttattgttcca atttggatcc 300 atnaatttgc nttcaattcc aggagctttg gaaggaattccaaggaaagc tccaggaaaa 360 ccgtattaaa ctttccaggg gccaaantcc ttcaccaattttttccacna actttccagg 420 cctgncncaa aaaaatggaa agggagttgg tangtccc 458<210> SEQ ID NO 12 <211> LENGTH: 388 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(11)..(12) <223> OTHER INFORMATION: n equals to a, t, g, or c <221>NAME/KEY: misc_feature <222> LOCATION: (46) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(50) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (81) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (138) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (155) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (182) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (188) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (269) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (317) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (322) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_difference <222> LOCATION: (358) <223> OTHER INFORMATION: n equalsto a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (363)<223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (375) <223> OTHER INFORMATION: n equals toa, t, g, or c <400> SEQUENCE: 12 ctgcactggg nncatgaact aggcctggccttcaccaaga accgantgan ctataccaac 60 aaattcctgc tgatcccaga ntcgggagactacttcattt actcccaggt cacattccgt 120 gggaatgaac ctctgaantg ccagtgaaaatcagncaagc aggccgacca aacaagccag 180 antccatnca ctgtggtcat caccaaggtaacagacagct accctgagcc aacccagctc 240 cttcatgggg accaagtttg tttgcgaantaggttagcaa ctggttccag cccattttac 300 cttgggggcc agttctnctt gncaagaaggggacaagctt atggtggaac gttcatanca 360 tcntttttgg gtggntttac acaaaagg 388<210> SEQ ID NO 13 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: TNF-gamma 5′primer with BamHI restriction site <400> SEQUENCE: 13 gcgcggatccaccatgagac gctttttaag caaagtc 37 <210> SEQ ID NO 14 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: TNF-gamma 3′ primer with XbaI restriction site <400>SEQUENCE: 14 cgcgtctaga ctatagtaag aaggctccaa agaagg 36 <210> SEQ ID NO15 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: TNF-gamma 5′ primer with BamHIrestriction site <400> SEQUENCE: 15 gcgcggatcc accatgagac gctttttaagcaaagtc 37 <210> SEQ ID NO 16 <211> LENGTH: 36 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:TNF-gamma 3′ primer with XbaI restriction site <400> SEQUENCE: 16cgcgtctaga ctatagtaag aaggctccaa agaagg 36 <210> SEQ ID NO 17 <211>LENGTH: 56 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: TNF-gamma 3′ primer containingsequences complementary to Xba I site, translation stop codon, and HAtag <400> SEQUENCE: 17 cgctctagat caagcgtagt ctgggacgtc gtatggatagtaagaaggct ccaaag 56 <210> SEQ ID NO 18 <211> LENGTH: 733 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 gggatccggagcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60 aattcgagggtgcaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120 tctcccggactcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg 180 tcaagttcaactggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240 aggagcagtacaacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300 ggctgaatggcaaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360 agaaaaccatctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420 catcccgggatgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480 atccaagcgacatcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540 ccacgcctcccgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcaggtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660 acaaccactacacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720 gactctagaggat 733 <210> SEQ ID NO 19 <211> LENGTH: 1116 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 19 atggccgagg atctgggactgagctttggg gaaacagcca gtgtggaaat gctgccagag 60 cacggcagct gcaggcccaaggccaggagc agcagcgcac gctgggctct cacctgctgc 120 ctggtgttgc tccccttccttgcaggactc accacatacc tgcttgtcag ccagctccgg 180 gcccagggag aggcctgtgtgcagttccag gctctaaaag gacaggagtt tgcaccttca 240 catcagcaag tttatgcacctcttagagca gacggagata agccaagggc acacctgaca 300 gttgtgagac aaactcccacacagcacttt aaaaatcagt tcccagctct gcactgggaa 360 catgaactag gcctggccttcaccaagaac cgaatgaact ataccaacaa attcctgctg 420 atcccagagt cgggagactacttcatttac tcccaggtca cattccgtgg gatgacctct 480 gagtgcagtg aaatcagacaagcaggccga ccaaacaagc cagactccat cactgtggtc 540 atcaccaagg taacagacagctaccctgag ccaacccagc tcctcatggg gaccaagtct 600 gtatgcgaag taggtagcaactggttccag cccatctacc tcggagccat gttctccttg 660 caagaagggg acaagctaatggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 720 gaagataaaa ccttctttggagccttctta ctataggagg agagcaaata tcattatatg 780 aaagtcctct gccaccgagttcctaatttt ctttgttcaa atgtaattat aaccaggggt 840 tttcttgggg ccgggagtaggggcattcca cagggacaac ggtttagcta tgaaatttgg 900 ggcccaaaat ttcacacttcatgtgcctta ctgatgagag tactaactgg aaaaaggctg 960 aagagagcaa atatattattaagatgggtt ggaggattgg cgagtttcta aatattaaga 1020 cactgatcac taaatgaatggatgatctac tcgggtcagg attgaaagag aaatatttca 1080 acaccttcct gctatacaatggtcaccagt ggtcca 1116 <210> SEQ ID NO 20 <211> LENGTH: 251 <212> TYPE:PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Met Ala Glu Asp LeuGly Leu Ser Phe Gly Glu Thr Ala Ser Val Glu 1 5 10 15 Met Leu Pro GluHis Gly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30 Ala Arg Trp AlaLeu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45 Gly Leu Thr ThrTyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60 Ala Cys Val GlnPhe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser 65 70 75 80 His Gln GlnVal Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 85 90 95 Ala His LeuThr Val Val Arg Gln Thr Pro Thr Gln His Phe Lys Asn 100 105 110 Gln PhePro Ala Leu His Trp Glu His Glu Leu Gly Leu Ala Phe Thr 115 120 125 LysAsn Arg Met Asn Tyr Thr Asn Lys Phe Leu Leu Ile Pro Glu Ser 130 135 140Gly Asp Tyr Phe Ile Tyr Ser Gln Val Thr Phe Arg Gly Met Thr Ser 145 150155 160 Glu Cys Ser Glu Ile Arg Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser165 170 175 Ile Thr Val Val Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu ProThr 180 185 190 Gln Leu Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly SerAsn Trp 195 200 205 Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu GlnGlu Gly Asp 210 215 220 Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu ValAsp Tyr Thr Lys 225 230 235 240 Glu Asp Lys Thr Phe Phe Gly Ala Phe LeuLeu 245 250 <210> SEQ ID NO 21 <211> LENGTH: 434 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (15) <223> OTHER INFORMATION: n equals to a, t, g, or c <221>NAME/KEY: misc_feature <222> LOCATION: (19) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(133) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (388) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (424) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 21tctacacaag gtacngacng ctaccctgag ccaacccagc tcctcatggg gaccaagtct 60gtatgcgaag taggtagcaa ctggttccag cccatctacc tcggagccat gttctccttg 120caagaagggg acnagctaat ggtgaacgtc agtgacatct ctttggtgga ttacacaaaa 180gaagataaaa ccttctttgg agccttctta ctataggagg agagcaaata tcattatatg 240aaagtcctct gccaccgagt tcctaatttt ctttgttcaa atgtaattat aaccaggggt 300tttcttgggg ccgggagtag ggggcattcc cacagggaca acggtttagc tatgaaattt 360ggggggccca aaatttcaca acttcatngt tgcccttact tgatgagaag tacttaactt 420gganaaaagg cttg 434 <210> SEQ ID NO 22 <211> LENGTH: 417 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (4) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (8) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (17) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (24) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (28) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (31)..(32) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (35) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (41)..(43) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (46) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (48) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (50) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (53) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (55) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (61)..(63) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (66)..(67) <223> OTHER INFORMATION: n equals to a, t, g,or c <221> NAME/KEY: misc_feature <222> LOCATION: (202) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (209) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (282) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (306) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (321) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (344) <223> OTHER INFORMATION: n equals to a, t, g, or c<221> NAME/KEY: misc_feature <222> LOCATION: (346) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (380)..(381) <223> OTHER INFORMATION: n equals to a, t,g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (395) <223> OTHERINFORMATION: n equals to a, t, g, or c <221> NAME/KEY: misc_feature<222> LOCATION: (405) <223> OTHER INFORMATION: n equals to a, t, g, or c<400> SEQUENCE: 22 attncggnac gagcagnggc atgnccgngg nnctnggactnnnctntngn gananagcca 60 nnnttnnaat gctgccagag cacggcagct gcaggcccaaggccaggagc agcagcgcac 120 gctgggctct cacctgctgc ctggtgttgc tccccttccttgcaggactc accacatacc 180 tgcttgtcag ccagcttcgg gnccagggng aggcctgtgtgcagttccag ggtctaaaag 240 gacaggagtt tgcaccttca catcagcaag tttatgcacctnttagagca gacggagata 300 agccangggg acaactgaca nttgtgagac aaattccacacagnanttta aaatcagttt 360 ccagttttga atggggacan nattaggctg gcttnacaagaccgntggat tttacag 417 <210> SEQ ID NO 23 <211> LENGTH: 388 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (11)..(12) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(46) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (50) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (81) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (138) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (155) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (182) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (188) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (269) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (317) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (322) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (358) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (363) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (375) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 23ctgcactggg nncatgaact aggcctggcc ttcaccaaga accgantgan ctataccaac 60aaattcctgc tgatcccaga ntcgggagac tacttcattt actcccaggt cacattccgt 120gggaatgaac ctctgaantg ccagtgaaaa tcagncaagc aggccgacca aacaagccag 180antccatnca ctgtggtcat caccaaggta acagacagct accctgagcc aacccagctc 240cttcatgggg accaagtttg tttgcgaant aggttagcaa ctggttccag cccattttac 300cttgggggcc agttctnctt gncaagaagg ggacaagctt atggtggaac gttcatanca 360tcntttttgg gtggntttac acaaaagg 388 <210> SEQ ID NO 24 <211> LENGTH: 458<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: misc_feature <222> LOCATION: (9) <223> OTHER INFORMATION: nequals to a, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION:(12) <223> OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (119) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (303) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (311) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (387) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (409) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (425) <223>OTHER INFORMATION: n equals to a, t, g, or c <221> NAME/KEY:misc_feature <222> LOCATION: (427) <223> OTHER INFORMATION: n equals toa, t, g, or c <221> NAME/KEY: misc_feature <222> LOCATION: (453) <223>OTHER INFORMATION: n equals to a, t, g, or c <400> SEQUENCE: 24ggcacagcng gnagtagggg gcattccaca gggacaacgg tttagctatg aaatttgggg 60cccaaaattt cacacttcat gtgccttact gatgagagta ctaactggaa aaaggctgna 120agagagcaaa tatattatta agatgggttg gaggattggc gagtttctaa atattaagac 180actggatcac tgaaatgaat ggatgatcta ctcgggtcca ggattgaaag agaaatattt 240caacaccttc ctgctataca atggtcacca gtggtccagt tattgttcca atttggatcc 300atnaatttgc nttcaattcc aggagctttg gaaggaattc caaggaaagc tccaggaaaa 360ccgtattaaa ctttccaggg gccaaantcc ttcaccaatt ttttccacna actttccagg 420cctgncncaa aaaaatggaa agggagttgg tangtccc 458 <210> SEQ ID NO 25 <211>LENGTH: 546 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: codon optimized form of TNF-gamma-beta<400> SEQUENCE: 25 atgctgaaag gtcaagaatt cgcaccgtcc caccagcaggtttacgcacc gctgcgtgca 60 gacggtgata agccgcgtgc acacctgacc gttgtgcgccagaccccgac ccagcacttc 120 aaaaaccagt tcccggctct gcactgggag cacgaactgggcctggcctt caccaagaac 180 cgcatgaact acaccaacaa attcctgctg atcccggagtctggtgacta cttcatctac 240 tcccaggtga ccttccgtgg tatgacctct gagtgctccgaaatccgtca ggcaggccgt 300 ccgaacaagc cggactccat caccgtggtg atcaccaaagtgaccgactc ttacccggag 360 ccgacccagc tgctgatggg taccaagtct gtttgcgaagttggttccaa ctggttccag 420 ccgatctacc tcggtgccat gttctccctg caagagggcgacaaactgat ggtgaacgtg 480 tccgacatct ctctggtgga ttacaccaag gaagataaaaccttcttcgg tgccttcctg 540 ctgtaa 546 <210> SEQ ID NO 26 <211> LENGTH:181 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: translation product of codon optimized form ofTNF-gamma-beta <400> SEQUENCE: 26 Met Leu Lys Gly Gln Glu Phe Ala ProSer His Gln Gln Val Tyr Ala 1 5 10 15 Pro Leu Arg Ala Asp Gly Asp LysPro Arg Ala His Leu Thr Val Val 20 25 30 Arg Gln Thr Pro Thr Gln His PheLys Asn Gln Phe Pro Ala Leu His 35 40 45 Trp Glu His Glu Leu Gly Leu AlaPhe Thr Lys Asn Arg Met Asn Tyr 50 55 60 Thr Asn Lys Phe Leu Leu Ile ProGlu Ser Gly Asp Tyr Phe Ile Tyr 65 70 75 80 Ser Gln Val Thr Phe Arg GlyMet Thr Ser Glu Cys Ser Glu Ile Arg 85 90 95 Gln Ala Gly Arg Pro Asn LysPro Asp Ser Ile Thr Val Val Ile Thr 100 105 110 Lys Val Thr Asp Ser TyrPro Glu Pro Thr Gln Leu Leu Met Gly Thr 115 120 125 Lys Ser Val Cys GluVal Gly Ser Asn Trp Phe Gln Pro Ile Tyr Leu 130 135 140 Gly Ala Met PheSer Leu Gln Glu Gly Asp Lys Leu Met Val Asn Val 145 150 155 160 Ser AspIle Ser Leu Val Asp Tyr Thr Lys Glu Asp Lys Thr Phe Phe 165 170 175 GlyAla Phe Leu Leu 180 <210> SEQ ID NO 27 <211> LENGTH: 182 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: 5′ primer to useful for generating 5′ half of codonoptimized form of TNF-gamma-beta <400> SEQUENCE: 27 ggaattccatatgctgaaag gtcaagaatt cgcaccgtcc caccagcagg tttacgcacc 60 gctgcgtgcagacggtgata agccgcgtgc acacctgacc gttgtgcgcc agaccccgac 120 ccagcacttcaaaaaccagt tcccggctct gcactgggag cacgaactgg gcctggcctt 180 ca 182 <210>SEQ ID NO 28 <211> LENGTH: 179 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 3′ primer touseful for generating 5′ half of codon optimized form of TNF-gamma-beta<400> SEQUENCE: 28 atcaccacgg tgatggagtc cggcttgttc ggacggcctgcctgacggat ttcggagcac 60 tcagaggtca taccacggaa ggtcacctgg gagtagatgaagtagtcacc agactccggg 120 atcagcagga atttgttggt gtagttcatg cggttcttggtgaaggccag gcccagttc 179 <210> SEQ ID NO 29 <211> LENGTH: 131 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: 5′ primer to useful for generating 3′ half of codonoptimized form of TNF-gamma-beta <400> SEQUENCE: 29 actccatcaccgtggtgatc accaaagtga ccgactctta cccggagccg acccagctgc 60 tgatgggtaccaagtctgtt tgcgaagttg gttccaactg gttccagccg atctacctcg 120 gtgccatgtt c131 <210> SEQ ID NO 30 <211> LENGTH: 135 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: 3′ primer touseful for generating 3′ half of codon optimized form of TNF-gamma-beta<400> SEQUENCE: 30 cgctctagat tattacagca ggaaggcacc gaagaaggttttatcttcct tggtgtaatc 60 caccagagag atgtcggaca cgttcaccat cagtttgtcgccctcttgca gggagaacat 120 ggcaccgagg tagat 135

What is claimed is:
 1. A method of treating or preventing aninflammatory disease or disorder comprising administering to an animalin which such treatment or prevention is desired an antibody or fragmentthereof that specifically binds TNF-gamma-beta protein in an amounteffective to treat or prevent the inflammatory disease or disorder. 2.The method of claim 1 wherein the animal is human.
 3. The method ofclaim 1 wherein the antibody or fragment thereof specifically binds aTNF-gamma-beta protein selected from the group consisting of: (a) aprotein whose sequence consists of amino acid residues 1 to 251 of SEQID NO:20; (b) a protein whose sequence consists of amino acid residues62 to 251 of SEQ ID NO:20; (c) a protein whose sequence consists ofamino acid residues 72 to 251 of SEQ ID NO:20; (d) a protein whosesequence consists of amino acid residues 101 to 251 of SEQ ID NO:20; (e)a protein whose sequence consists of the amino acid sequence of thefull-length polypeptide encoded by the cDNA contained in ATCC DepositNumber 203055; (f) a protein whose sequence consists of the amino acidsequence of the extracellular domain of the polypeptide encoded by thecDNA contained in ATCC Deposit Number 203055; and (g) a protein whosesequence consists of the amino acid sequence of the mature form of thepolypeptide encoded by the cDNA contained in ATCC Deposit Number 203055.4. The method of claim 3 wherein the antibody or fragment thereof is amonoclonal antibody.
 5. The method of claim 3 wherein the antibody orfragment thereof is a human antibody.
 6. The method of claim 3 whereinthe antibody or fragment thereof is a humanized antibody.
 7. The methodof claim 3 wherein the inflammatory disease or disorder is inflammatorybowel disease.
 8. The method of claim 3 wherein the inflammatory diseaseor disorder is encephalitis.
 9. The method of claim 3 wherein theinflammatory disease or disorder is atherosclerosis.
 10. The method ofclaim 3 wherein the inflammatory disease or disorder is psoriasis.
 11. Amethod of treating or preventing inflammation comprising administeringto an animal in which such treatment or prevention is desired anantibody or fragment thereof that specifically binds TNF-gamma-betaprotein in an amount effective to treat or prevent the inflammation. 12.The method of claim 11 wherein the animal is a human.
 13. The method ofclaim 11 wherein the antibody specifically binds a TNF-gamma-betaprotein selected from the group consisting of: (a) a protein whosesequence consists of amino acid residues 1 to 251 of SEQ ID NO:20; (b) aprotein whose sequence consists of amino acid residues 62 to 251 of SEQID NO:20; (c) a protein whose sequence consists of amino acid residues72 to 251 of SEQ ID NO:20; (d) a protein whose sequence consists ofamino acid residues 101 to 251 of SEQ ID NO:20; (e) a protein whosesequence consists of the amino acid sequence of the full-lengthpolypeptide encoded by the cDNA contained in ATCC Deposit Number 203055;(f) a protein whose sequence consists of the amino acid sequence of theextracellular domain of the polypeptide encoded by the cDNA contained inATCC Deposit Number 203055; and (g) a protein whose sequence consists ofthe amino acid sequence of the mature form of the polypeptide encoded bythe cDNA contained in ATCC Deposit Number
 203055. 14. The method ofclaim 13 wherein the antibody or fragment thereof is a monoclonalantibody.
 15. The method of claim 13 wherein the antibody or fragmentthereof is a human antibody.
 16. The method of claim 13 wherein theantibody or fragment thereof is a humanized antibody.
 17. A method oftreating or preventing an autoimmune disease or disorder comprisingadministering to an animal in which such treatment or prevention isdesired an antibody or fragment thereof that specifically bindsTNF-gamma-beta protein in an amount effective to treat or prevent theautoimmune disease or disorder.
 18. The method of claim 17 wherein theanimal is human.
 19. The method of claim 17 wherein the antibody orfragment thereof specifically binds a TNF-gamma-beta protein selectedfrom the group consisting of: (a) a protein whose sequence consists ofamino acid residues 1 to 251 of SEQ ID NO:20; (b) a protein whosesequence consists of amino acid residues 62 to 251 of SEQ ID NO:20; (c)a protein whose sequence consists of amino acid residues 72 to 251 ofSEQ ID NO:20; (d) a protein whose sequence consists of amino acidresidues 101 to 251 of SEQ ID NO:20; (e) a protein whose sequenceconsists of the amino acid sequence of the full-length polypeptideencoded by the cDNA contained in ATCC Deposit Number 203055; (f) aprotein whose sequence consists of the amino acid sequence of theextracellular domain of the polypeptide encoded by the cDNA contained inATCC Deposit Number 203055; and (g) a protein whose sequence consists ofthe amino acid sequence of the mature form of the polypeptide encoded bythe cDNA contained in ATCC Deposit Number
 203055. 20. The method ofclaim 19 wherein the antibody or fragment thereof is a monoclonalantibody.
 21. The method of claim 19 wherein the antibody or fragmentthereof is a human antibody.
 22. The method of claim 19 wherein theantibody or fragment thereof is a humanized antibody.
 23. The method ofclaim 19 wherein the autoimmune disease or disorder is systemic lupuserythematosus.
 24. The method of claim 19 wherein the autoimmune diseaseor disorder is arthritis.
 25. The method of claim 24 wherein theautoimmune disease or disorder is rheumatoid arthritis.
 26. The methodof claim 19 wherein the autoimmune disease or disorder is multiplesclerosis.
 27. The method of claim 19 wherein the autoimmune disease ordisorder is Crohn's disease.
 28. The method of claim 19 wherein theautoimmune disease or disorder is autoimmune encephalitis.
 29. A methodof treating or preventing graft versus host disease (GVHD) comprisingadministering to an animal in which such treatment or prevention isdesired an antibody or fragment thereof that specifically bindsTNF-gamma-beta protein in an amount effective to treat or prevent theGVHD.
 30. The method of claim 29 wherein the animal is a human
 31. Themethod of claim 29 wherein the antibody or fragment thereof specificallybinds a TNF-gamma-beta polypeptide selected from the group consistingof: (a) a protein whose sequence consists of amino acid residues 1 to251 of SEQ ID NO:20; (b) a protein whose sequence consists of amino acidresidues 62 to 251 of SEQ ID NO:20; (c) a protein whose sequenceconsists of amino acid residues 72 to 251 of SEQ ID NO:20; (d) a proteinwhose sequence consists of amino acid residues 101 to 251 of SEQ IDNO:20; (e) a protein whose sequence consists of the amino acid sequenceof the full-length polypeptide encoded by the cDNA contained in ATCCDeposit Number 203055; (f) a protein whose sequence consists of theamino acid sequence of the extracellular domain of the polypeptideencoded by the cDNA contained in ATCC Deposit Number 203055; and (g) aprotein whose sequence consists of the amino acid sequence of the matureform of the polypeptide encoded by the cDNA contained in ATCC DepositNumber
 203055. 32. The method of claim 31 wherein the antibody orfragment thereof is a monoclonal antibody.
 33. The method of claim 31wherein the antibody or fragment thereof is a human antibody.
 34. Themethod of claim 31 wherein the antibody or fragment thereof is ahumanized antibody.
 35. A method of killing a cell of hematopoieticorigin comprising contacting a TNF-gamma-beta protein with a cell ofhematopoietic origin.
 36. The method of claim 35 wherein the TNF-gammabeta protein is selected from the group consisting of: (a) a proteinwhose sequence consists of amino acid residues 1 to 251 of SEQ ID NO:20;(b) a protein whose sequence consists of amino acid residues 62 to 251of SEQ ID NO:20; (c) a protein whose sequence consists of amino acidresidues 72 to 251 of SEQ ID NO:20; (d) a protein whose sequenceconsists of amino acid residues 101 to 251 of SEQ ID NO:20; (e) aprotein whose sequence consists of a fragment of at least 30 contiguousamino acid residues of the polypeptide of SEQ ID NO:20 that hasTNF-gamma-beta functional activity. (f) a protein whose sequenceconsists of the amino acid sequence of the full-length polypeptideencoded by the cDNA contained in ATCC Deposit Number 203055; (g) aprotein whose sequence consists of the amino acid sequence of theextracellular domain of the polypeptide encoded by the cDNA contained inATCC Deposit Number 203055; (h) a protein whose sequence consists of theamino acid sequence of the mature form of the polypeptide encoded by thecDNA contained in ATCC Deposit Number 203055; and (i) a protein whosesequence consists a fragment of at least 30 contiguous amino acidresidues of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 203055 that has TNF-gamma-beta functional activity. 37.The method of claim 36 wherein the TNF-gamma-beta protein isradiolabeled.
 38. The method of claim 37 wherein the radiolabel isselected from the group consisting of: (a) ¹²⁵I; (b) ¹³¹I; (c) ¹¹¹In;(d) ⁹⁹Tc; (e) ¹⁷⁷Lu; (f) ⁹⁰Y; (g) ¹⁶⁶Ho; and (h) ¹⁵³Sm.
 39. The methodof claim 36 wherein the TNF-gamma-beta protein is conjugated to acytotoxic agent or cytotoxic pro-drug.
 40. The method of claim 36wherein the cell of hematopoietic origin is a T cell.
 41. The method ofclaim 40 wherein the T cell is cancerous.
 42. A method of killing a cellof hematopoietic origin comprising administering to an animal in whichsuch killing of hematopoietic cells is desired, a TNF-gamma-beta proteinin an amount effective to kill the cell.
 43. The method of claim 42wherein the animal is a human.
 44. The method of claim 42 wherein theTNF-gamma-beta protein is selected from the group consisting of: (a) aprotein whose sequence consists of amino acid residues 1 to 251 of SEQID NO:20; (b) a protein whose sequence consists of amino acid residues62 to 251 of SEQ ID NO:20; (c) a protein whose sequence consists ofamino acid residues 72 to 251 of SEQ ID NO:20; (d) a protein whosesequence consists of amino acid residues 101 to 251 of SEQ ID NO:20; (e)a protein whose sequence consists of a fragment of at least 30contiguous amino acid residues of the polypeptide of SEQ ID NO:20 thathas TNF-gamma-beta functional activity. (f) a protein whose sequenceconsists of the amino acid sequence of the full-length polypeptideencoded by the cDNA contained in ATCC Deposit Number 203055; (g) aprotein whose sequence consists of the amino acid sequence of theextracellular domain of the polypeptide encoded by the cDNA contained inATCC Deposit Number 203055; (h) a protein whose sequence consists of theamino acid sequence of the mature form of the polypeptide encoded by thecDNA contained in ATCC Deposit Number 203055; and (i) a protein whosesequence consists a fragment of at least 30 contiguous amino acidresidues of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 203055 that has TNF-gamma-beta functional activity. 45.The method of claim 44 wherein the TNF-gamma-beta protein isradiolabeled.
 46. The method of claim (a) wherein the radiolabel isselected from the group consisting of: (i) ¹²⁵I; (k) ¹³¹In; (l) ⁹⁹Tc;(m) ¹⁷⁷Lu; (n) ⁹⁰Y; (o) ¹⁶⁶Ho; and (p) ¹⁵³Sm.
 47. The method of claim 44wherein the TNF-gamma-beta protein is conjugated to a cytotoxic agent orcytotoxic pro-drug.
 48. The method of claim 44 wherein the cell ofhematopoietic origin is a T cell.
 49. The method of claim 48 wherein theT cell is cancerous.